Microluminometer chip and method to measure bioluminescence

ABSTRACT

An integrated microluminometer includes an integrated circuit chip having at least one n-well/p-substrate junction photodetector for converting light received into a photocurrent, and a detector on the chip for processing the photocurrent. A distributed electrode configuration including a plurality of spaced apart electrodes disposed on an active region of the photodetector is preferably used to raise efficiency.

The present application is a divisional application of U.S. patentapplication Ser. No. 09/660,581 filed Sep. 12, 2000, now U.S. Pat. No.6,905,834 issued Jun. 14, 2005, said application being acontinuation-in-part application of U.S. Ser. No. 08/978,439 filed Nov.25, 1997, now U.S. Pat. No. 6,117,643 issued Sep. 12, 2000, the entirecontents of which are specifically incorporated herein by reference inits entirety.

The United States government has rights in the present inventionpursuant to grant number DE-FG05-94ER61870 from the Department of Energyand grant number F49620-89-C-0023 from the United States Air Force.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Electronic circuitry may be used to detect a luminescent response. Inparticular, one may use an optical application specific integratedcircuit (OASIC), which combines analog signal conditioning, digitalsignal processing, and wireless transmission with a sensitiveelectro-optical detector. To achieve maximum sensitivity to theluminescent response of a bioreporter, an OASIC should be sensitive tolight in the 400 nm to 700 nm (visible) range, should have low leakagecurrent and low noise, and should have minimal sensitivity to changes inenvironmental factors such as temperature and humidity. Such devices maybe manufactured via a standard complimentary-metal-oxide-semiconductor(CMOS) process on a single substrate.

2. Description of Related Art

A bioluminescent bioreporter is an organism that is geneticallyengineered to produce light when a particular substance is metabolized.For example, bioluminescent (lux) transcriptional gene fusions may beused to develop light emitting reporter bacterial strains that are ableto sense the presence, bioavailability, and biodegradation of organicchemical pollutants such as naphthalene, toluene, and isopropylbenzene.In general, the lux reporter genes are placed under regulatory controlof inducible degradative operons maintained in native or vector plasmidsor integrated into the chromosome of the host strain.

Due to the widespread use of petroleum products and the currentregulations requiring underground storage tanks to be upgraded, replacedor closed by December 1998, the number of petroleum-contaminated siteshas abounded. Of particular concern for drinking water quality are themore water-soluble components, benzene, toluene, ethylbenzene andxylenes (BTEX). Natural attenuation which relies on in situbiodegradation of pollutants has received a large amount of attentionespecially for petroleum contaminants. While microorganisms capable ofbiodegradation of BTEX compounds are usually present at these sites,there is a need to know whether or not conditions are favorable forbiodegradation to occur.

Bioluminescent reporters have been widely used for the real timenon-destructive monitoring of gene expression. Heitzer et al. (1992)developed a quantitative assay for naphthalene bioavailability andbiodegradation using a nah-lux reporter strain HK44 constructed by Kinget al. (1990) containing a lux transposon (Tn4431) insertion in nahG ofthe lower naphthalene degradation operon. The nah-lux reporter wasexpanded for use as an on-line optical biosensor for application ingroundwater monitoring (Heitzer et al., 1994). Other lux fusions havebeen constructed for monitoring the expression of catabolic genesincluding those for degradation of isopropylbenzene (Selifonova et al.,1996) and toluene (Applegate et al., 1997).

In addition to catabolic gene fusions, a wide variety of genes andoperons have been studied using lux fusions. Lux fusions have beenconstructed for monitoring heat shock genes expression, oxidativestress, presence of Hg(II) and alginate production. In all these cases,the lux fusions are plasmid-based and were constructed by placing thepromoter of interest in front of the promoterless lux genes from Vibriofischeri contained in pUCD615 (Rogowsky et al., 1987).

3. Deficiencies in the Prior Art

A need has arisen for a monolithic bioelectronic device that containsboth a bioreporter and an OASIC, yet is very small, rugged, inexpensive,low power, and wireless. (A monolithic bioelectronic device is a devicethat contains biological and electrical components and that isconstructed on a single substrate layer.) Such a bioluminescentbioreporter integrated circuit (BBIC) could detect substances such aspollutants, explosives, and heavy-metals residing in inhospitable areassuch as groundwater, industrial process vessels, and battlefields.Applications for such a device include environmental pollutantdetection, oil exploration, drug discovery, industrial process control,and hazardous chemical monitoring. The low cost of such sensors and thewide variety of deployment methods would allow a large number of them tobe distributed over a wide area for very comprehensive coverage.

SUMMARY OF THE INVENTION

The invention concerns analyte sensing devices comprising engineeredbioluminescent bacteria placed on an integrated microluminometer. Thebacteria are engineered to luminesce when a targeted compound isdetected or metabolized. The microluminometer detects, processes, andmeasures the magnitude of the optical signal.

In certain embodiments, the invention discloses an apparatus fordetecting the concentration of a selected substance or analyte in asample. The apparatus generally comprises an integrated circuit thatincludes a phototransducer operative to generate a signal in response tolight, a container for holding bioluminescent bacteria, and a substratethat is attached to the container and to the integrated circuit. Theanalyte concentration is related to the light signal.

Within the context of the present invention, bioluminescent bacteria maybe referred to as bioreporters or bioreporter molecules because of theirresponse to a selected analyte by expressing a luminescent lux geneproduct.

The apparatus may further comprise a layer of bioresistant/biocompatiblematerial between the substrate and the container, such as a layer ofsilicon nitride. The integrated circuit is preferably a CMOS integratedcircuit, and the phototransducer is preferably a photodiode. Theintegrated circuit may also include a current to frequency converterand/or a digital counter. Additionally, the integrated circuit may alsoinclude one or more transmitters. Such transmitters may be wireless, orconventionally wired. In other embodiments, the apparatus also includesa central data collection station capable of receiving transmissionsfrom the transmitter.

The apparatus may also contain one or more fluid or nutrient reservoirsand one or more microfluidic pumps on the substrate to provide nutrientmeans for the bioreporter organisms utilized with the apparatus. Anexemplary bioreporter is a genetically engineered bacterium, yeast, oranimal cell.

The selectively permeable container may comprise a polymer matrix, whichallows gas or fluid to reach the bioreporter. Preferably, the matrix isoptically-clear. Optionally, the integrated circuit may contain a globalpositioning system (GPS). The BBIC may be prepared in a housing (e.g.,injection molded plastic) that permits free passage of the gas orliquid, yet blocks ambient light. Such a housing may comprise aflat-black finish and a maze-like passage-way. The fluid or gas easilytraverses the turns in the passageway, while the ambient light isgreatly attenuated (due to the flat-black finish) at each turn.

An additional embodiment of the invention is an apparatus that detects asubstance, such as a fluid comprising an integrated circuit including aphototransducer adapted to input an electrical signal into the circuitin response to light, a bioreporter that metabolizes the substance andemitting light consequent to such metabolism, the reporter adapted tocontact the substance; and a transparent, biocompatible, andbioresistant separator positioned between the phototransducer and thebioreporter to enable light emitted from the bioreporter to strike thephototransducer. The bioreporter may be a bacterium, fungal, yeast,plant, or animal cell, or alternatively, a nucleotide sequence whichencodes a luminescent reporter molecule. The apparatus may also comprisea plastic matrix encasing the bioreporter and enabling contact betweenthe substance and the bioreporter. Such a matrix may be permeable to thesubstance.

Another aspect of the invention is an apparatus for detecting theconcentration of a particular substance, comprising a substrate, aluminescent microorganism such as Pseudomonas fluorescens HK44 thatmetabolizes a selected substance to emit light; a selectively permeablecontainer affixed to the substrate capable of holding the luminescentmicroorganism and which allows gas or fluid to reach the bioreporter,and prevents ambient light from reaching the bioreporter; a layer ofsemiconducting material such as silicon nitride between the substrateand the container; a fluid and nutrient reservoir and microfluidic pumpon the substrate; a Complementary Metal Oxide Semiconductor (CMOS)integrated circuit on the substrate including a photodiode operative togenerate a current in response to the light, a current to frequencyconverter, a digital counter, and wireless transmitter; and, a centraldata collection station capable of receiving transmissions from thetransmitter.

Yet another aspect of the invention concerns a monolithic bioelectronicdevice for detecting a substance in a sample. This device generallycomprises a bioreporter capable of metabolizing the substance andemitting light consequent to such metabolism; and, a sensor capable ofgenerating an electrical signal in response to the reception of theemitted light. Such a device may also include a transparent,bioresistant and biocompatible separator positioned between thebioreporter and the sensor.

A standard integrated circuit (IC) is coated with a layer of insulatingmaterial such as silicon dioxide or silicon nitride. This process iscalled passivation and serves to protect the surface of the chip frommoisture, contamination, and mechanical damage. Although this coating isadequate for general purpose chips, BBICs may be used in a variety ofpossibly harsh environments for which the standard passivation processis inadequate. For these purposes, BBICs require a second coating thatmust be biocompatible and bioresistant, must protect the OASIC fromchemical stresses, must be optically tuned to efficiently transmit thelight from the material under test, must adhere to an oxide coating,must be pin-hole free, and must be able to be patterned in order to formopenings over the bonding pads and whatever structures that might beneeded to maintain the bioreporter or collect a sample.

While the individual components of the invention described herein may beobtained and assembled individually, the inventors contemplate that, forconvenience, the components of the biosensor may be packaged in kitform. Kits may comprise, in suitable container means, one or morebioreporters and an integrated circuit including a phototransducer. Thekit may comprise a single container means that contains one or morebioreporters and the integrated circuit including a phototransducer.Alternatively, the kits of the invention may comprise distinct containermeans for each component. In such cases, one container would contain oneor more bioreporters, either pre-encapsulated or encapsulated in anappropriate medium disclosed herein, and another container would includethe integrated circuit. When the bioreporter is pre-encapsulated, thekit may contain one or more encapsulation media. The use of distinctcontainer means for each component would allow for the modulation ofvarious components of the kits. For example, several bioreporters may beavailable to chose from, depending on the substance one wishes todetect. By replacing the bioreporter, one may be able to utilize theremaining components of the kit for an entirely different purpose, thusallowing reuse of components.

The container means may be a container such as a vial, test tube,packet, sleeve, shrink-wrap, or other container means, into which thecomponents of the kit may be placed. The bioreporter also may bealiquoted into smaller containers, should this be desired.

The kits of the present invention also may include a means forcontaining the individual containers in close confinement for commercialsale, such as, e.g., injection or blow-molded plastic containers intowhich the desired vials are retained.

Irrespective of the number of containers, the kits of the invention alsomay comprise, or be packaged with, an instrument for assisting with theplacement of the bioreporter upon the integrated circuit. Such aninstrument may be a syringe, pipette, forceps, or any other similardevice.

In fact, virtually any suitable packaging and delivery of the requiredcomponents are contemplated to be useful so long as the bioreporterremains functional. For example, long term storage of a bioreporter chipon a matrix such as alginate and a bacterial suspension may requirerefrigeration and prevention of desiccation for the organisms to remainviable.

The kit may comprise one or more distinct bioreporters and a singlephotodetector. For example, a kit for detecting naphthalene and toluenein a sample might comprise one bioreporter and a biofilm for detectingnaphthalene and a distinct and/or separate biofilm for detectingtoluene. The two biofilms may be applied sequentially to the sensor witheach compound tested separately, or in certain circumstances, thebiofilms may be applied in tandem to the chip and the compounds testedsimultaneously. Various examples are given in FIG. 9A, FIG. 9B, FIG. 9C,and FIG. 9D. Several biofilms may be placed in an array as shown in FIG.9A, allowing several different bioreporters to be tested simultaneously.The inventors contemplate that the number of distinct biofilms may beinfinite, provided that the signal produced by a single individualbiofilm is detectable by the photodetector. Alternatively, as shown inFIG. 9B, each distinct biofilm may be applied sequentially to the chip.Furthermore, as shown in FIG. 9C, several bioreporters may be mixedwithin one or more biofilms. Also, the biofilms may be layered as inFIG. 9D to allow several biofilms to be measured simultaneously.

In a certain particular embodiments, the invention includes biosensorsfor the detection of ammonia, generally determined as ammonium ion. Insuch examples, a bioreporter microorganism is situated close to thesurface of an integrated circuit chip, generally in a semipermeablecontainer or matrix, so that in the presence of an ammonium ion, themicroorganism produces a luminescent protein that emits light related tothe amount of ammonium ion present. The microorganism is engineered toinclude lux genes stably integrated into the chromosome which arecontrolled by promoters responsive to ammonia. Examples are the hau oramo promoters, although it is contemplated that other promotersresponsive to ammonia or ammonium ion are also suitable. Exemplary luxgenes are the CDABC lux gene fusions that can be prepared for examplefrom lux genes of bacteria such as Vibrio fischeri.

Exemplary bacteria into which the lux gene construct may be introducedinclude E. coli, Salmonella, Mycobacter tuberculosis, Listeria,Photobacter phosphoreum or Vibrio fischeri.

The invention also includes expression vectors comprising lux CDABEgenes operatively linked to a promoter induced by ammonia or ammoniaion, cells transformed by such vectors and any of a number of apparatuscomprising the described biosensor. Additionally, the invention is alsointended to include methods for detecting ammonia using the biosensorand apparatus described and kits including the biosensors. Such kitswill include instructions for use as well as optional materials such asstandards for preparing standard curves.

A further exemplary biosensor has been designed to detect estrogens andxenoestrogens for use in eukaryotic cells such as yeast.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1 shows a perspective view of one embodiment of the presentinvention.

FIG. 2 Shows the measured signal that resulted from a test of aprototype of the present invention.

FIG. 3A shows a high-quality photodetector that can be made using astandard N-well CMOS process.

FIG. 3B shows two photodetector structures fabricated in asilicon-on-insulator CMOS process: on the left, a lateral PIN detector;on the right a device similar to left except that the junction is formedwith a Schottky junction.

FIG. 4 shows the photodetector in FIG. 1 together with associated signalconditioning and processing circuitry on a single integrated circuit.

FIG. 5 shows a block diagram of one possible embodiment of the signalprocessing portion of the present invention.

FIG. 6 shows a side view of one embodiment of the present invention.

FIG. 7A shows a simple photodiode consisting of a P-diffusion layer, anN-well, and a P-substrate.

FIG. 7B shows a circuit using a large area photodiode for efficientlight collection, and a small-area diode in a feedback loop to supplythe forward bias current that cancels out the photo-current.

FIG. 7C shows a circuit using correlated double sampling (CDS) tominimize the effects of low frequency (flicker) amplifier noise as wellas time or temperature dependent variations in the amplifier offsetvoltage.

FIG. 8 shows a bioreporter being supplied with water and nutrients.

FIG. 9A, FIG. 9B, FIG. 9C and FIG. 9D show conceptual diagrams depictingmethods of utilizing multiple bioreporters. Different bioreporters aresymbolized by A, B, C, etc.

FIG. 9A shows a biofilm separated into a number of discreet sectionswith each section comprising a different bioreporter.

FIG. 9B shows a number of biofilms, each comprising a differentbioreporter.

FIG. 9C shows multiple bioreporters combined within a single biofilm.

FIG. 9D shows a biofilm comprising several discreet layers with eachlayer comprising a different bioreporter.

FIG. 10 shows the sequence of primers used in site-directed mutagenesisto generate the modified mini-Tn5 and the cloning vector, pLJS.Asterisks denote mismatches between the primer and the target sequence.*A* denotes an extra adenine which was inadvertently synthesized.

FIG. 11 shows a diagram for the construction of the mini-Tn5 Kmtod-lux.A/X and Nh/X represent AvrII-XbaI and NheI-XbaI heterologous cloningsites, respectively. Abbreviations: N, NotI; Sa, Sal I, X, XbaI.

FIG. 12 shows the cloning plasmid pLJS with unique restriction sites.Abbreviations: A, AvrII; Ac, Acc I; Ap, Apa I; B, Bam HI; Bs, Bst XI; C,Cla I; D, Dra II; E, Eco RI; Ea, Eag I; EV, Eco RV; H, HindIII; Hc, HincII; K, Kpn I; N, NotI; Nh, NheI; P, Pst I; S, SpeI; Sa, Sal I; Sc, SacI; Sc II, Sac II, Sm, SmaI; X, XbaI, Xh, Xho I.

FIG. 13 shows the bioluminescence response of TVA8 to increasingconcentrations of toluene after 2 h exposure. Values are averages ofthree replicates and have been normalized to the cell density (OD₅₄₆).

FIG. 14 shows growth curves for batch cultures of TVA8 (circles) and F1(triangles) grown on MSM with toluene vapor. Values are averages ofthree replicates and error bars represent one standard deviation.

FIG. 15 shows a bioluminescence and growth of TVA8 on toluene vaporunder batch conditions. O, □, Δ represent individual replicates ofbioluminescence readings over time. The closed squares represent theaverage optical density at 546 nm (OD₅₄₆) of three replicates.

FIG. 16 shows the construction of the tod-lux reporter plasmid pUTK30.The 2.75-kb EcoR1-XbaI fragment from pDTG514 (Menn et al., 1991) wascloned in front of the promoterless lux gene cassette in pUCD615(Rogowsky et al., 1987). Abbreviations: B, BamHI; Gb, BglII; E, EcoR1;H, HindIII; K, KpmI; Ps, PstI; Pv, PvuII; Sc, SacI; S, SalI; Sm, SmaI;X, XbaI.

FIG. 17 shows a diagram of the on-line DVR system used to monitor theco-metabolism of TCE.

FIG. 18 shows bioluminescent response to varying concentrations oftoluene (λ) and JP4 jet fuel, expressed as mg L⁻¹ toluene (σ) in growingcell assays after a 1.5-h exposure.

FIG. 19 shows bioluminescent response to multiple and single exposuresof 10 mg L⁻¹ toluene by resting cells of P. putida B2 in batch studies.Symbols: μ multiple exposure; Δ single exposure.

FIG. 20 shows bioluminescence and co-metabolism of TCE by P. putida B2in response to square wave perturbations of 10 mg L⁻¹ toluene in 20-hcycles. Symbols: λ bioluminescence; σ, TCE in effluent; ν, toluene ineffluent; ----, TCE in feed; ---, toluene in feed.

FIG. 21 shows exploded, cutaway diagram of the reactor. Feed isdistributed to the reactor cavity filled with cells immobilized in smallalginate beads by channels etched in the reactor body and by theattached metal flit. An annular insert holds the 0.2 μM hydrophobicfilter against the top metal flit with the effect of providing asignificant uniform resistance to flow and providing a clean effluentfor automatic injection into the HPLC. The resistance to flow caused bythe filter was typically 50 psig for a clean filter.

FIG. 22 shows absorption isotherms of naphthalene and sodium salicylateon calcium alginate. Naphthalene adsorbed linearly at experimentalconditions, whereas salicylate did not appreciably partition.

FIG. 23 shows actual and predicted concentrations of studies 1 a-c and 4a-f. Error bars are shown with average values. The solid line representsthe model predictions using the least-squares reaction rate constant forthe complete data set. The model is overall second order, first order inbiomass and first order in salicylate, with a rate constant of 2.23×10⁻²dm³/g mol. The empirical data depicted are from Table 8.

FIG. 24 shows actual and predicted concentrations of studies 2 a-c and 3a-f. Error bars are shown with data points. The solid line representsthe model predictions using the least-squares reaction rate constant forthe complete data set. The model is overall second order, first order inbiomass and first order in salicylate, with a rate constant of 2.23×10⁻²dm³/g mol. The empirical data depicted are from Table 8.

FIG. 25 show an unusual transient response was observed when a clean bedof HK44 was “shocked” by the step addition of salicylate. The transientresponse may be caused by an initial imbalance resulting from the rapidtransport of the inducer into the cell and an initial slow rate ofdegradation. After this initial transient behavior, light intensitymimicked the concentration of inducer. This transient behavior was onlyobserved at the beginning of the study. Light intensity trackedsubsequent changes in inducer concentration.

FIG. 26 shows specific steady-state light emission byalginate-immobilized P. fluorescens HK44 as a function of estimatedconcentration inside the PBR at the light probe. Standard deviations areshown with the average values. The lines represent the average linearresponse for each data set.

FIG. 27 shows the response of HK44 to salicylate in a flow cell. Lightintensity mimicked the rise and fall of salicylate concentration in theflow cell. HK44 was immobilized in alginate on a photodiode.

FIG. 28 shows the response of HK44 to naphthalene in a flow cell. Lightintensity mimicked the rise and fall of naphthalene concentration in theflow cell. HK44 was immobilized in alginate on a photodiode. A largerlag in response was observed than in FIG. 27. The lag times may resultfrom the way that naphthalene and salicylate are transported into thecell and consumed. Physical processes such as adsorption also have aneffect on lag time.

FIG. 29A and FIG. 29B show normalized logarithmic light levels within 5h of induction. Light levels are expressed in nA cfu⁻¹. No data areshown for the groundwater at pH 3-5, as no light was produced. YPEGrepresents yeast extract/peptone/glucose medium.

FIG. 29A shows response due to induction with simple solution, SS.

FIG. 29B shows response due to induction with complex solution, CS.

FIG. 30A and FIG. 30B show percentage salicylate uptake by immobilizedHK44.

FIG. 30A shows uptake following induction with SS.

FIG. 30B shows uptake following induction with CS.

FIG. 31 shows operation of HK44 in alginate beads. The logarithm of thenumber of colony-forming units/alginate beads is shown.

FIG. 32 shows rates of the bioluminescence reaction with SS and CS. Thenormalized rates were calculated from the set of light data collectedwithin the 5 h post-induction period. This set of data was used in thecalculation of the regression covariance.

FIG. 33 shows IC mounted on a common honeybee as part of Oak RidgeNational Laboratory research on micro-transmitters.

FIG. 34 shows BBICs connected together in a distributed neural network

FIG. 35 shows a single BBIC from the distributed neural network.

FIG. 36 shows a bioluminescent bioreporter integrated circuit formed byplacing genetically-engineered bioluminescent cells on anoptically-sensitive integrated circuit (IC). The molecular specificityis provided by the cells, while the IC provides the advantages of amicroelectronic format.

FIG. 37 shows a BBIC with a microluminometer for detectingbioluminescence and other integrated sensors (e.g. temperature sensor),information processing circuitry, and wireless telemetry.

FIG. 38 shows two of the semiconductor junctions available forphotodiode realization in bulk, n-well, CMOS IC processes. Ann-well/substrate junction provides higher quantum efficiency.

FIG. 39 shows two photodiode electrode configurations: (a) the n-wellelectrode covers most of the active area of the photodiode; and (b)distributed n-well electrodes reduce leakage current and detectorcapacitance with an insignificant effect on the quantum efficiency.

FIG. 40 illustrates a typical photodiode electrode configuration forBBIC applications. The n-wells are 5.6 μm×5.6 μm and thecenter-to-center spacing is 12.6 μm. The wiring grid above the Sisurface is shown. Connection to the n-well electrodes is made at theintersection of the vertical and horizontal wires.

FIG. 41 is a graph showing reverse leakage current vs. reverse bias at40° C., 42.5° C., and 45° C. for the photodiode of FIG. 40 illustratingthe advantage of operating the photodiode at low reverse bias.

FIG. 42 is a graph of photocurrent vs. reverse bias at input fluxes of1.6×10⁷, 2.6×10⁷, 5.5×10⁷, and 1.8×10⁸ photons/sec. illustrating thatthe quantum efficiency is only slightly influenced by the magnitude ofthe reverse bias above 50 mV.

FIG. 43 shows a gated integrator that integrates the photocurrent from0≦t≦t₀ forms the causal portion of the matched filter for dcluminescence in wide-band white noise.

FIG. 44 shows a current-to-frequency converter used to form the longtime-constant integrator.

FIG. 45 shows a microluminometer that measures 2.2 mm×2.2 mm.

FIG. 46 shows the bioluminescence response for a culture containingvarious concentrations of P. fluorescens 5RL cells. Bioluminescence wasdetermined using the integrated circuit luminometer and a light-tightenclosure mounted above the chip. Linear regression analysis showed thatthe data fit a linear model indicating that bioluminescence per cellremains constant. Using this linear model, the limit of detection forthis experimental configuration was calculated to be 4×10⁵ cells per mL.

FIG. 47 shows bioluminescence as a function of time for a culturecontaining 4×10⁸ CFU/mL. The results show a dramatic decrease in thebioluminescence with time, possibly due to oxygen limitation caused bythe quiescent conditions of the vial.

FIG. 48 is a comparison of bioluminescence signal as detected by theintegrated circuit and a PMT-based luminometer.

FIG. 49 shows a Mini-Tn5 containing the hao-lux fusion.

FIG. 50 shows a slot blot analysis of eight selected Kn^(r) N. europaeaclones using the lux gene as a probe.

FIG. 51 shows a growth curve of N. europaea ATCC19178 (control, amo-luxand hao-lux fusions). A: optical density measurements at 600 nm; and B:nitrite production.

FIG. 52 shows light emission (photons/s) (A) and light/OD (B) of N.europaea ATCC19178 (control), amo-lux and hao-lux fusions.

FIG. 53 shows bioluminescent response of N. Europaea Km^(r) hao-lux toincreasing concentration of (NH)₂SO₄ after 30, 60 and 90 min exposure.

FIG. 54 shows the response of sol-gel encapsulated Saccharomycescerevisiae HER to β-estradiol.

FIG. 55 shows BBIC photodetector design.

FIG. 56 shows a photodetector leakage current vs. reverse bias andtemperature.

FIG. 57 shows a photodetector leakage current vs. reverse bias andtemperature.

FIG. 58 shows a photosignal vs. reverse bias at four input flux levels.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The preferred embodiments of the present invention are illustrated inFIGS. 1-32 of the drawings, like numerals being used to refer to likeand corresponding parts of the various drawings.

Genetically engineered bioluminescent bacteria are encapsulated andplaced on a specially designed integrated circuit. The bacteria aredesigned to bioluminesce after metabolizing a targeted analyte, whilethe integrated circuit is designed to detect the luminescence, processthis signal, and report the results. Exemplary bioreporters aredescribed including one for the sensing ammonia and another forestrogens. Methods for adhering these bioreporters to integratedcircuits within encapsulation matrices are described as well as severaldesign features of the integrated circuit that improve performance andsensitivity.

Overview of the System

A photodiode is integrated into a semiconductor substrate along withsignal processing electronics and either data storage electronics,electronics for transmission of the measured data via a hard-wiredcommunication network or wireless communication electronics for remoteread-out of the data. Key elements of the micro-luminometer system are aphotodiode compatible with the semiconductor process employed tofabricate the accompanying electronics, novel low-noise electronics forthe detection of low-level photosignals in the presence of electronicnoise and communications electronics (wired or wireless) to transmit thedata to a data processing and storage system.

FIG. 1 shows a perspective view of the present invention. The substance20 that is being detected enters the BBIC 21 through the polymer matrix22. Once the substance is detected, the BBIC transmits a signalindicating the concentration of the substance to a central location.

FIG. 6 shows a side view of the present invention. The bioreporter isenclosed in polymer matrix 103, which is separated from a photodetector102 by a protective coating 101. A single substrate 100 contains theseelements as well as additional circuitry 104 that processes andtransmits the signal.

FIG. 3A shows a high-quality photodetector made using a standard N-wellCMOS process. The photodetector consists of two reverse biased diodes inparallel. The top diode is formed between the P+ active layer 45 and theN-well 46, and the bottom diode is formed between the N-well 46 and theP-substrate 47. The top diode has good short wavelength lightsensitivity (400-550 nm), while the bottom diode provides good longwavelength sensitivity (500-1100 nm). Thus, the complete diode issensitive over the range from 400 to 1100 nm. The luminescent compoundunder test 41 is separated from the photodetector by a layer 40 of Si₃N₄and a layer 42 of SiO₂.

FIG. 4 shows the photodetector 66 in FIG. 1 coupled with signalconditioning 65 and processing circuitry 64 on a single integratedcircuit 60. The purpose of the analog signal conditioning circuitry isto amplify and filter the relatively small photodetector signal so thatit can be compared to a threshold, digitized, or modulated fortransmission. While the effects of wideband noise can be reduced byintegration of the signal, integration has a much weaker effect on 1/fnoise. The effect of low frequency noise can be reduced by usingcorrelated double sampling (CDS) in which two samples are taken within ashort interval of time such that one sample consists of signal and noiseand the other sample consists only of noise. The low frequency componentof the noise is greatly attenuated in the difference of these twosamples.

When the targeted substance reaches the bioreporter, it is metabolizedand the bioreporter emits light with a wavelength of from between about400 and about 700 nm (in the visible range). The bioreporter is encasedin a polymer matrix that keeps the bioreporter positioned over thephotodetector, allows the gas or fluid being sampled to reach thebioreporter, and allows the emitted light to reach the photodetector.

A block diagram showing one possible embodiment of the signal processingportion of the present invention is shown in FIG. 5. The photodetectorin FIG. 3A is a photodiode 81 that responds to light by conducting acurrent to the ground. A current to frequency converter 82 converts thiscurrent into a sequence of pulses that are counted by a digital counter83. The number of pulses counted in a fixed period of time is directlyproportional to the amount of light collected by the photodiode, whichin turn is directly proportional to the concentration of the targetedsubstance. A wireless transmitter 84 then relays this measuredconcentration 85 to a central data collection station.

FIG. 8 shows the bioreporter being supplied with water and nutrients. Afluid and nutrient reservoir 141 is connected to a microfluidic pump 142so that nutrient and fluid 144 may flow through the polymer matrix 143enclosing the bioreporter. Each of these components can be constructedon a single substrate 140.

A prototype device was constructed by coupling P. fluorescens HK44, anaphthalene bioreporter, to an OASIC. The resulting device was exposedto naphthalene. The measured signal is shown in FIG. 2. The backgroundreading is indicated from 0 to 10 min, and the reading during inducedbioluminescence is shown from 10 to 20 min.

Additional circuitry may be included in the BBIC as required. Forexample, a BBIC may contain a Global Positioning Satellite system fordetermining the location of the sensor.

Photodetector

The first element in the micro-luminometer signal processing chain isthe photodetector. The key requirements of the photodetector are:

-   -   Sensitivity to wavelength of light emitted by the bioluminescent        or chemiluminescent compound under test;    -   Low background signal (i.e., leakage current) due to parasitic        reverse biased diodes;    -   Appropriate coating to prevent the materials in the        semiconductor devices from interfering with the bioluminescent        or chemiluminescent process under study and to prevent the        process under study from degrading the performance of the        micro-luminometer; and,    -   Compatibility with the fabrication process used to create the        microluminometer circuitry.

Two photodetector configurations that satisfy these requirements aredescribed below. It should be understood, however, that alternativemethods of constructing such a photodetector may be used by one skilledin the art without departing from the spirit and scope of the inventionas defined in the claims.

In the first embodiment, the photodetector is fabricated in a standardN-well CMOS process. Shown in FIG. 3A, this detector is formed byconnecting the PN junction between the PMOS active region and the N-wellin parallel with the PN junction between the N-well and the P-typesubstrate. The resulting detector is sensitive to light between about400 nm and about 1100 nm, a range that encompasses the 450-600 nmemission range of most commonly used bioluminescent and chemiluminescentcompounds or organisms. In order to meet the requirement that the devicehave a low background signal, the device is operated with a zero bias,setting the operating voltage of the diode equal to the substratevoltage. The photodiode coating may be formed with a deposited siliconnitride layer or other material compatible with semiconductor processingtechniques.

In the second photodetector embodiment, the detector is fabricated in asilicon-on-insulator (SOI) CMOS process. The internal leakage current inan SOI process is two to three orders of magnitude lower than instandard CMOS due to the presence of a buried oxide insulating layerbetween the active layer and the substrate. Two photodetector structuresare envisioned in the SOI process. The first structure, shown on theleft of FIG. 3B, consists of a lateral PIN detector where the P-layer isformed by the P+ contact layer, the I (intrinsic) region is formed bythe lightly doped active layer, and the N region is formed by the N+contact layer of the SOI CMOS process. The spectral sensitivity of thislateral detector is set by the thickness of the active layer, which maybe tuned for specific bioluminescent and chemiluminescent compounds.

The second structure, shown on the right side of FIG. 3B, is similar tothe first except that the junction is formed with a Schottky junctionbetween a deposited cobalt silicide (CoSi₂) or other appropriatematerial layer and the lightly doped active layer.

The inventors contemplate that other photodetector configurations may beenvisioned in silicon or other semiconductor processes meeting thecriteria set forth above.

Low Noise Electronics

The low noise electronics are the second element in themicro-luminometer signal processing chain. The requirements for the lownoise electronics are:

-   -   Sensitivity to very low signal levels provided by the        photodetector;    -   Immunity to or compensation for electronic noise in the signal        processing chain;    -   Minimum sensitivity to variations in temperature;    -   Minimum sensitivity to changes in power supply voltages (for        battery powered applications);    -   For some applications the electronics must have sufficient        linearity and dynamic range to accurately record the detected        signal level; and,    -   In other applications the electronics must simply detect the        presence of a signal even in the presence of electronic and        environmental noise.

Three embodiments that satisfy these requirements are considered below.It should be understood, however, that alternative methods of detectingsmall signals while satisfying these requirements can be used withoutdeparting from the spirit and scope of the invention as defined in theclaims.

FIG. 7A schematically shows the first approach to the detection of verysmall signals. This device uses a P-diffusion/N-well photodiode, astructure compatible with standard CMOS IC processes, in the opencircuit mode with a read-out amplifier (fabricated on the same IC withthe photodiode). The luminescent signal generates electron-hole pairs inthe P-diffusion and the N-well. The photo-generated electrons in theP-diffusion are injected into the N-well, while the photo-generatedholes in the N-well are injected into the P-diffusion. The N-well istied to ground potential so that no charge builds up in this region.However, since the P-diffusion is only attached to the input impedanceof a CMOS amplifier (which approaches infinity at low frequencies), apositive charge collects in this region. Thus, the voltage on theP-diffusion node begins to rise.

As the P-diffusion voltage begins to rise, the P-diffusion/N-wellphotodiode becomes forward biased, thereby producing a current in adirection opposite to the photo-generated current. The system reachessteady state when the voltage on the P-diffusion node creates a forwardbias current exactly equal in magnitude (but opposite in polarity) tothe photo-current. If this PN junction has no deviations from the idealdiode equation, then the output voltage isV _(out) =V _(t)ln(I _(p)/(AI _(s))+1),  (1)where V_(t) is the thermal voltage (approximately 26 mV at roomtemperature), I_(p) is the photo-current, A is the cross-sectional areaof this PN junction, and I_(s) is the reverse saturation current for aPN junction with unit cross-sectional area. The value of I_(s) dependsgreatly on the IC process and material parameters.

Two major error currents are present in PN junctions operating at lowcurrent density: recombination current and generation current. Except atvery low temperatures, free carriers are randomly created in the PNjunction space charge region. Since this region has a high field, thesethermally excited carriers are immediately swept across the junction andform a current component (generation current) in the same direction asthe photocurrent. Carriers crossing the space-charge region also have afinite chance of recombining. This creates another current component(recombination current) in the opposite direction of the photocurrent.Therefore, taking into account these error currents, equation (1)becomesV _(Out) =V _(t)ln((I _(p) +I _(g) −I _(r))/(A I _(s))+1)  (2)This output voltage is a function of parameters that are generallybeyond our control. However, we do have control over the junction area,A. Unfortunately, to make our output signal larger, we want a small A,while we want a large A for a high quantum efficiency (QE).

FIG. 7B shows a second microluminometer embodiment that satisfies bothof these needs. This circuit uses a large area photodiode for efficientlight collection, but uses a small-area diode in a feedback loop tosupply the forward bias current that cancels out the photo-current. Onceagain, the amplifier and feedback diodes are fabricated on the same ICas the photodiode. For this circuit,V _(out)=3 V _(t) ln((I _(p) +I _(g) −I _(r))/(A _(fb) I _(s))+1),  (3)where A_(fb) is the small cross-sectional area of the feedback diode.More than one diode is used in the feedback path to make the outputsignal large compared to the DC offset of any subsequent amplifierstages. This technique allows efficient collection of the light with alarge-area photodiode, yet produces a large output voltage because ofthe small-area diodes in the feedback path.

The feedback circuit of FIG. 7B maintains the photodiode at zero bias.With no applied potential, the recombination and generation currentsshould cancel. Equation (3) becomesV _(out)=3 V _(t) ln((I _(p)/(A _(fb) I _(s)))+1)  (4)if the smaller recombination and generation currents in the smallerfeedback diodes are neglected.

The principal advantages of the second micro-luminometer embodimentshown in FIG. 7B are:

-   -   The SNR is totally determined by the photodiode. Noise from the        small diode and amplifier are negligible;    -   Diodes can be added in the feedback path until the signal level        at the output of the amplifier is significant compared to offset        voltages (and offset voltage drift) of subsequent stages;    -   This method is completely compatible with standard CMOS        processes with no additional masks, materials, or fabrication        steps;    -   This detection scheme can be fabricated on the same IC with        analog and digital signal processing circuits and RF        communication circuits; and,    -   Measurement can be made without power applied to the circuit.        Power must be applied before the measurement can be read, but        the measurement can be obtained with no power.

A third micro-luminometer implementation shown in FIG. 7C usescorrelated double sampling (CDS) to minimize the effects of lowfrequency (flicker) amplifier noise as well as time or temperaturedependent variations in the amplifier offset voltage. As shown in FIG.7C, a photodiode with capacitance C_(d) and noise power spectral densityS_(i) is connected to an integrating preamplifier with feedbackcapacitance C_(f) and input noise power spectral density S_(v) through aset of switches that are controlled by the logical level of a flip-flopoutput. When the flip-flop output is low, the switches are positioned sothat the photocurrent flows out of the preamplifier, causing the outputvoltage of the integrator to increase. When the low-pass filteredintegrator output voltage exceeds a threshold, V_(HI), the uppercomparator “fires,” setting the flip-flop and causing its output to gohigh. The detector switches change positions, causing current to flowinto the integrating amplifier, which in turn causes the amplifieroutput voltage to decrease. When the integrator output goes below asecond threshold, V_(LO), the lower comparator “fires,” resetting theflip-flop and causing the output to go low again. The process repeatsitself as long as a photocurrent is present.

The average period of the output pulse, Δt, is given by $\begin{matrix}{{{\Delta\quad t} = \frac{2{C_{f}\left( {V_{HI} - V_{LO}} \right)}}{I_{p}}},} & (5)\end{matrix}$where V_(HI) and V_(LO) are the threshold voltages of the comparatorsand I_(p) is the diode photocurrent. Two noise sources contribute toerror in the measured value of Δt. S_(i) is the input noise currentpower spectral density associated primarily with the photodiode, andS_(v) is the input noise voltage power spectral density associatedprimarily with the preamplifier. The diode noise is given by$\begin{matrix}{{S_{i} = {2{q\left( {{2I_{s}} + I_{p}} \right)}\left( \frac{A^{2}}{H\quad z} \right)}},} & (6)\end{matrix}$where I_(s) is the photodiode reverse saturation current and I_(p) isthe photocurrent. As the photocurrent approaches zero, the noise powerspectral density approaches a finite value of 4qI_(s) A²/Hz. The noisevoltage S_(v) of the preamplifier is determined by its design and hasunits of V²/Hz.

The transfer function from the point where the diode noise is introducedto the output of the integrator is given approximately by$\begin{matrix}{{{H_{i}(\omega)} \approx {\left( \frac{1}{{sC}_{f}} \right)\left( \frac{\omega_{1}}{s + \omega_{1}} \right)}},} & (7)\end{matrix}$where □₁ is the corner frequency of the integrating amplifier and s=j□.Ignoring for the moment the effect of the switches, the transferfunction from the point where the amplifier noise is introduced to theoutput of the integrator is given approximately by $\begin{matrix}{{{H_{v}(\omega)} \approx {\left( \frac{C_{f} + C_{d}}{C_{f}} \right)\left( \frac{\omega_{1}}{s + \omega_{1}} \right)}},} & (8)\end{matrix}$

The switches perform a correlated double sampling function whichattenuates the noise which appears below the switching frequency of theoutput pulse string. The transfer function of a correlated doublesampling circuit is approximated to first order by the expression$\begin{matrix}{{{H(\omega)} \approx \left( \frac{s}{s + {{2/\Delta}\quad t}} \right)},} & (9)\end{matrix}$where Δt is the average period of the output pulse string. Thus, takinginto account the switches, the transfer function from the point wherethe amplifier noise is introduced to the output of the integrator isapproximately given by $\begin{matrix}{{H_{v}(\omega)} \approx {\left( \frac{C_{f} + C_{d}}{C_{f}} \right)\left( \frac{\omega_{1}}{s + \omega_{1}} \right){\left( \frac{s}{s + {2/\overset{\_}{\Delta\quad t}}} \right).}}} & (10)\end{matrix}$

This is an important result because the effective zero introduced in thenoise voltage transfer function reduces the effect of the flicker noiseof the amplifier. This is particularly useful in CMOS implementations ofthe micro-luminometer where flicker noise can have a dominant effect.

The mean squared output noise at the output of the integrator is$\begin{matrix}{{v_{n}^{2} = {{\int_{- \infty}^{\infty}{S_{v}\left( {H_{v}*H_{v}} \right)}} + {{S_{i}\left( {H_{i}*H_{i}} \right)}{\mathbb{d}\omega}}}},} & (11)\end{matrix}$

and the RMS noise voltage is then given byσ_(ν)=√{square root over (ν _(n) ²)}.  (12)

The RMS error in the measured period is determined by the slope of theintegrated signal and the noise at the output of the integratorfollowing the relationship $\begin{matrix}{\sigma_{t} = \frac{\sigma_{v}}{\frac{\mathbb{d}v}{\mathbb{d}t}}} & (13)\end{matrix}$

or, approximately, $\begin{matrix}{\sigma_{t} \approx {\frac{\sigma_{v}}{\frac{\left( {V_{HI} - V_{LO}} \right)}{\Delta\quad t}}.}} & (14)\end{matrix}$

The error in measuring Δt may be reduced by collecting many outputpulses and obtaining an average period. The error in the measuredaverage pulse period improves proportionately to the square root of thenumber of pulses collected, such that $\begin{matrix}{{{\overset{\_}{\sigma}}_{t} \approx {\frac{\sigma_{v}}{\frac{\left( {V_{HI} - V_{LO}} \right)}{\Delta\quad t}}\frac{1}{\sqrt{N}}}}{or}} & (15) \\{{\overset{\_}{\sigma}}_{t} \approx {\frac{\sigma_{v}}{\frac{\left( {V_{HI} - V_{LO}} \right)}{\Delta\quad t}}\sqrt{\frac{t_{meas}}{\Delta\quad t}}}} & (16)\end{matrix}$

where t_(meas) is the total measurement time.

Thus, implementation of the micro-luminometer has the followingadvantages:

-   -   The low frequency “flicker” noise of the amplifier is reduced by        a correlated double sampling process; and,    -   Ideally, the accuracy of the measured photocurrent may be        improved without limit by acquiring data for increasing periods        of time.

Of course, practical limitations imposed by the lifetime and stabilityof the signals produced by the luminescent compound under test willultimately determine the resolution of this implementation.

Read-Out Electronics

Several methods of reading out the data from the micro-luminometer maybe used. These include:

-   -   Generation of a DC voltage level proportional to the        photocurrent;    -   Generation of a DC current level proportional to the        photocurrent;    -   Generation of a logical pulse string whose rate is proportional        to the photocurrent;    -   On-chip implementation of an analog to digital converter that        reports a numerical value proportional to the photocurrent;    -   On-chip implementation of a serial or parallel communications        port that reports a number proportional to the photocurrent;    -   Implementation of an on-chip wireless communication system that        reports the value of the photocurrent;    -   Generation of a logical flag when the photocurrent exceeds a        predefined level; and,    -   Generation of a radio-frequency signal or beacon when the        photocurrent exceeds a predefined level.        Biosensors for Chemical and Biological Agents

A BBIC requires integration of the appropriate bioreporters, a cellentrapment method, an integrated microluminometer, and aviocompatible/bioresistant protective coating for the integrated chip.

Luminometry

Luminometry is an analytical technique that uses chemiluminescence orbioluminescence to detect the presence and concentration of a particularsubstance, condition, or organism. Luminescent assays provide one of themost important and widely used analytical tools now in use. For example,luciferase bioluminescence assays are used to quantify the amount ofadenosine triphosphate (ATP) present in samples. This type ofmeasurement indicates the presence or absence of small numbers ofmicrobes, and is useful for environmental toxindetection/quantification, water quality measurements, as well as manyother applications. Similar bioluminescent techniques have been used tostudy gene expression (Bronstein, et al., 1994), detect heavy metal andorganic environmental pollutants (Applegate, et al., 1996), medicaldiagnostics or bioluminescent assays for research in auditoryneuroscience studies (Wangemann, 1996).

Luminometry is often the analytical technique of choice due to its highsensitivity and is routinely used to detect picogram levels of ATP.There is no requirement for an excitation source, and no radioactivematerials are involved. Luminescence is typically measured by bench-topluminometers which use photomultiplier tubes (PMTs), microchannelplates, or films as detection devices. While extremely sensitive, thesedevices are limited to laboratory use because of size, fragility, andcost. Applications that require in situ, in vivo, in vitro, or a largenumber of distributed or parallel measurements are not well served bypresent state-of-the-art luminometers. There is a need to makeluminescence-based assays rugged and inexpensive tools that operate in avariety of environments outside the laboratory.

Attributes of the integrated CMOS microluminometer include minimumdetectable signal (MDS) and immunity from false signals generated bythermally induced leakage current variations. These attributes aredetermined by the material characteristics, biasing, and front-endsignal processing of the CMOS photodiodes used to detect theluminescence.

CMOS Microluminometer Design

Applications for BBICs include environmental monitoring, food and waterquality testing, in vivo sensors for disease detection and management,and other remote applications where size, power consumption, and cableplant concerns are the dominant issues. Therefore, the integratedcircuit (IC) portion of the BBIC should reside on a single chip, becompatible with battery operation, and be compatible with RF circuitsfor wireless telemetry in addition to allowing the integration ofhigh-quality photodiodes and low-noise analog signal processing. FIG. 36shows a bioluminescent bioreporter integrated circuit formed by placinggenetically-engineered bioluminescent cells on an optically sensitiveintegrated circuit (IC). The molecular specificity is provided by thecells, while the IC provides the advantages of a microelectronic format.FIG. 37 shows a block diagram of one embodiment of the IC portion of aBBIC.

A standard 0.5-μm bulk CMOS process that meets optical and signalprocessing requirements, while allowing the realization of RF circuitsoperating in the 916-MHz band may be used. The design and performance ofthe two major components of the microluminometer; the CMOS photodiodesand the front-end signal processing, are described.

An integrated CMOS microluminometer for the detection of low-levelbioluminescence in whole-cell biosensing applications has beendeveloped. The microluminometer is the microelectronic portion of thebioluminescent bioreporter integrated circuit (BBIC). This device usesthe n-well/p-substrate junction of a standard bulk CMOS IC process toform the integrated photodetector. The photodetector uses a distributedelectrode configuration that minimizes detector noise. Signal processingis accomplished with a current-to-frequency converter circuit that formsthe causal portion of the matched filter for dc luminescence inwide-band white noise. Measurements show that luminescence can bedetected from as few as 4×10⁵ cells/mL.

Size, power consumption, and cable plant concerns are important in manyapplications. Ideally, the integrated circuit (IC) portion of the BBICresides on a single chip, is compatible with battery operation, andallows flexible communications with central data collection stations inaddition to accommodating the integration of high-quality photodiodesand low-noise analog signal processing. A standard 0.5-μm bulk CMOSprocess that meets optical and signal processing requirements wasselected which provided the desired size and power attributes. Thedesign and performance of the two major components of themicroluminometer: the CMOS photodiodes and the front-end signalprocessing are described.

CMOS Photodiodes

CMOS technology allows the realization of phototransistors, photodiodes,and photogates without any modification or additions to the standardprocessing steps. As normally used, these devices have broad spectralresponsivities that peak in the red/near infrared region. Peak externalquantum efficiency of 50%-80% has been reported for CMOS photodiodes(Kramer, et al., 1992).

FIG. 38 shows two junctions are available for the realization of CMOSphotodiodes in standard CMOS processes: p-diff/n-well andn-well/substrate. The shallower junction (p-diff/n-well) would seem tobe the most attractive for this application since its response peaksnear the 490-nm wavelength of the bioluminescence, yet drops off quicklyat longer wavelengths (Simpson, et al., 1998).

The quantum efficiency of p-diff/n-well photodiodes in small geometryCMOS processes is low (typically less than 10%). One explanation is thatthe shorter drive-in diffusion step for small geometry processes isinsufficient to anneal the lattice damage created by the ionimplantation step, thereby leaving a high density of charge traps inthese diffusions. In this case, the large number of charge carrier trapsseverely degrades the quantum efficiency in the blue and green opticalregimes. Regardless of the mechanism, the p-diff/n-well junction is notsuitable for low-level luminescence detection, prompting selection ofthe n-well/substrate photodiode for the microluminometer transducer.

The physical layout of the electrodes affects both the quantumefficiency and the reverse leakage current of the photodiode. Twopossible electrode configurations are shown in FIG. 39. In the firstconfiguration, the n-well electrode covered the entire active region ofthe photodiode. The advantage of this approach is that allphoto-generated charge is produced in the n-well and must only diffuse ashort distance to the n-well/substrate junction without being trapped toproduce a photocurrent.

The second approach employs an array of small n-well/substrate junctionsspread across the active region of the detector. This approach minimizesthe degradation of noise performance caused by detector capacitance andleakage current. However, in this configuration charge created in thesubstrate regions must diffuse a relatively long distance without beingtrapped to produce a photocurrent. In principle one could calculate theoptimum spacing between electrodes given a detailed knowledge ofmaterial parameters such as the diffusion length and the surfacerecombination velocity. These parameters may vary from run to run sothat empirical determination of optimum spacing may be preferred. As anexample, an initial choice of 5.6 μm×5.6 μm electrodes spaced 12.6 μmapart (˜20% coverage) was made as shown in FIG. 40.

For use with bioluminescent bioreporters, it is desirable to minimizethe photodiode reverse leakage current for two reasons. First, the powerspectral density of the detector white noise depends directly on themagnitude of the dc leakage current. Possibly more important is theinability to distinguish a low-level dc luminescent signal from a dcleakage current. Variations in the leakage current as a function oftemperature cannot be distinguished from a change in thebioluminescence. Conventional solutions, such as chopping the opticalsignal, are not practical for this integrated, single-chip, analyticalinstrument. The ideal diode equation, $\begin{matrix}{I_{f} = {I_{s}\left( {{\mathbb{e}}^{\frac{V_{f}}{V_{T}}} - 1} \right)}} & (17)\end{matrix}$where

-   -   I_(f)=forward current    -   I_(s)=reverse saturation current    -   V_(f)=forward bias    -   V_(T)=thermal voltage (≈26 mV@room temperature)        describes two competing current components: 1) electrons/holes        on the n/p side overcoming the potential barrier;        $\begin{matrix}        {{I_{f} = {I_{s}{\mathbb{e}}^{\frac{V_{f}}{V_{T}}}}},} & (18)        \end{matrix}$        and 2) holes/electrons on the n/p side diffusing to the edge of        the space charge region and being swept across        I _(r) =−I _(s).  (19)        At zero bias these two components are in dc equilibrium, so the        dc leakage current is zero. However, these currents are        uncorrelated, so their noise power spectral densities (PSD) add.        This simple analysis predicts that the noise PSD at zero bias is        higher than it is at any reverse bias.

Unfortunately, the situation is not that simple. Equation (17) describesmoderate to strong forward bias current. However, at weak forward biasor in reverse bias, equation (17) underpredicts the magnitude of thecurrent because of surface and generation/recombination effects. I_(r).as well as I_(f). will depend on bias, and it is not certain at whatbias level the minimum noise is found. However, zero bias is certainlywhere the minimum dc leakage current is found, and therefore thegreatest immunity from thermally generated false signals.

FIG. 41 shows the reverse leakage current vs. bias for the photodiode ofFIG. 40 at three different temperatures. This figure clearly shows thatoperating at reduced bias greatly reduces the magnitude of thetemperature drift of the leakage current. FIG. 42 shows the measuredphotodiode signal (minimum input flux=1.6×10⁷ photons/second,wavelength=490 nm) vs. reverse bias for the photodetector shown in FIG.40. This figure demonstrates that the quantum efficiency has a weakdependence on bias for reverse biases above 50 mV. In addition, thisfigure shows the quantum efficiency of this detector to be ˜70% at 490nm at 1.75 pA photocurrent for an input flux of 1.6×photons/sec, whichindicates that the spacing between n-well electrodes can be increased,thereby further decreasing leakage current and detector capacitance.

Signal Processing

The simplest noise approximation for the microluminometer assumes thedetection of a dc signal in wide band white noise. If the input signalx(t) is approximated as a step function u(t), then the impulse responseof the matched filter is:h _(opt)(t)=ku(t ₀ −t),  (20)where k is a constant and t₀ is the time of the measurement. The optimalimpulse response has an output at negative infinity for an impulse inputat t=0, and is therefore non-causal and non-realizable. However, thecausal portion of the filter can be realized as a gated integrator withthe gate open for 0<t<t₀ (FIG. 43).

The noise at the output of a gated integrator due to white detectorcurrent noise at the input is: $\begin{matrix}{{\overset{\_}{v_{no}^{2}} = \frac{\overset{\_}{i_{n}^{2}t_{0}}}{2C_{f}^{2}}},} & (21)\end{matrix}$where

-   -   {overscore (ν² _(no))}=mean square output voltage noise    -   {overscore (i_(n) ²)}=mean square photodiode current noise    -   C_(f)=integrator feedback capacitor,        while $\begin{matrix}        {{{v_{0}^{2}\left( t_{0} \right)} = \frac{i_{p}^{2}t_{0}^{2}}{C_{f}^{2}}},} & (22)        \end{matrix}$        where    -   ν_(o) ² (t_(o))=output signal power at t₀    -   i_(p)=photocurrent.        From equations (22) and (23) the signal-to-noise ration (SNR)        is: $\begin{matrix}        {{{SNR} = \frac{2i_{p}^{2}t_{0}}{i_{n}^{2}}},} & (23)        \end{matrix}$        and continues to improve as to increases.

Practical concerns generally limit t₀ to several minutes. A remainingproblem is capacitor values that are too large for on-chipimplementation. This was solved by using a hybrid analog/digitalintegration scheme as shown in FIG. 44. In this circuit, an analogintegrator and a discriminator convert the photodiode current into atrain of digital pulses (current-to-frequency converter (CFC)). Thesepulses are counted for a fixed time (t₀), and the result is a digitalword that is proportional to the photocurrent. This scheme has severaladvantages compared to other processing options including fast recoveryfrom overload and ease of analog-to digital conversion. It has beenreported as useful in optical detection systems (deGraff andWolffenbuttel, 1997).

Integrated CMOS Microluminometer

The disclosed sensors provide the basis for creating wholly selfcontained biosensors that require no exogenous reagents beyond what canbe provided on the IC so that the IC can function independently of anyother instrumental component

Microluminometer Chip

FIG. 45 shows a photograph of the complete microluminometer chip. Thechip measures 2.2 mm×2.2 mm with the photodetector occupying ˜25% (1.2mm²—although the total active region of the photodiode may be muchlarger) of the total chip area. For testing purposes the chip wasmounted in a 40-pin ceramic dual inline package.

Nutrient Delivery System

FIG. 21 illustrates one method of providing nutrients to the livingbioreporters on BBICs. The concept has three main components:

a fluid and nutrient reservoir;

a microfluidic pump; and,

a BBIC.

The fluid and nutrient reservoir and microfluidic pump on a differentsubstrate than the BBIC may be easier to implement. However, animplementation that places all three components on the same monolithicsubstrate could also be used.

The reservoir is simply a container that holds water with theappropriate nutrients in solution. This can be implemented on-chip bydepositing a thick oxide over the fluidic area of the chip and defininga reservoir space by photolithographic methods. To increase the volumeof such an implementation, an external container (e.g., a plasticpipette tip) may be attached to the on-chip reservoir with anappropriate epoxy.

Microfluidic pumps have been realized in numerous manners includingperistaltic pumps, conducting polymer pumps, and electro-osmotic pumps.For an on-chip pump, an electro-osmotic pump is most compatible. Thisdevice consists of a capillary that has been etched into the Si and thencoated with a thermally-grown oxide. A top plate is required for properpump operation. Polydimethylsiloxane (PDMS), sold under the brand nameSylgard 184, (Dow Corning) may be used to coat the top plate. Glass orquartz slides may also be used to form the top plate. The capillarycould be tens of microns wide and tens of microns in depth. The lengthcan be several centimeters, but on a BBIC would likely be on the orderof a few mm. To activate the pump, a voltage is placed across thecapillary. In capillary electrophoresis applications, voltages as highas 1 kV are required for rapid separations. However, in thisapplication, we would expect operation at only a few volts.

A gravity pump could be also be used where the floor of the capillary isat a slant. The end of the capillary that supplies the fluid to thebioreporters could be restricted to regulate the flow of fluid or anactuator (e.g., a micro-cantilever) could gate fluid flow. In practice,any pump that is small, low power, and can operate from low voltagescould be used either on-chip or on a separate substrate.

Bioluminescence Detection

Bioluminescence was determined for cultures containing differentconcentrations of P. fluorescens 5RL cells growing in LB supplementedwith 10 ppm of the inducer molecule salicylate and 14.7 mg/Ltetracycline (FIG. 46). Bioluminescence was determined using theintegrated circuit microluminometer and a light-tight enclosure mountedabove the chip. Linear regression analysis showed that the data fit alinear model indicating that bioluminescence per cell remains constantfor cell concentration ranging from 4×10⁵ to 2×10⁸ CFU/mL and fordetector responses ranging from 0.05 to 20 pA (FIG. 46). Using a linearmodel, the limit of detection for this experimental geometry wasestimated to be 4×10⁵ cells per mL. At cell concentrations greater than4×10⁸ CFU/mL, the bioluminescence decreased, possibly due to oxygenlimitation caused by the quiescent conditions of the vial (FIG. 47).

The results obtained with the BBIC microluminometer were compared withresults collected with the Azur PMT-based luminometer at each cellconcentration (FIG. 48). The data showed that the measuredbioluminescence responses were proportional for cell concentrationsranging 4×10⁵ to 2×10⁸ CFU/mL, indicating that the BBIC microluminometergave consistent results to standard PMT-based detection systems.

Biosensors

A “biosensor” generally refers to a small, portable, analytical devicebased on the combination of recognition biomolecules with an appropriatetransducer, and which detects chemical or biological materialsselectively and with high sensitivity. A treatise on this subject isgiven by Paddle (1996), from which the following is excerpted:

They may be used to detect toxic substances from a variety of sourcessuch as air, water or soil samples or may be used to monitor enclosedenvironments. They also may be formulated as catheters for monitoringdrug and metabolite levels in vivo, or as probes for the analysis oftoxic substances, drugs or metabolites in samples of say, blood andurine. Some biosensors with these potentials are currently eithercommercially available or undergoing commercial development(Alvarez-Icaza and Bilitewski, 1993).

A great number of review articles (e.g., Grate et al., 1993) and severalbooks (e.g., Hall, 1991) have been written describing both thetheoretical and practical aspects of individual biosensor technologiesand their development.

Biosensors

In biosensors, different biological elements may be combined withvarious kinds of transducers provided that the reaction of thebiological element with the substrate can be monitored. Table 1 liststhe transducer types available and biological elements that have beencombined with them to form a biosensor (Griffiths and Hall, 1993).

Biological Component of Biosensors

The biological components of biosensors are not only responsible for theselective recognition of the analyte, but also the generation of thephysiochemical signal monitored on the transducer and, ultimately, thesensitivity of the final device. They can be divided into two distinctcategories: catalytic and non-catalytic. The catalytic group includesenzymes, microorganisms and tissues. Devices incorporating theseelements are appropriate for monitoring metabolites in the millimolar tomicromolar range and can be used for continuous monitoring. Thenon-catalytic or affinity class biological component comprisesantibodies (or antigens), lectins, receptors and nucleic acids which aremore applicable to ‘single use’ disposable devices for measuringhormones, steroids, drugs, microbial toxins, cancer markers and virusesat concentrations in the micromolar to picomolar range. More recently, ahybrid configuration of biosensor has been introduced which combines theattributes of both the high affinity (‘irreversible’) binding of anantibody or DNA/RNA probe with the amplification characteristics of anenzyme. These systems are capable of monitoring analytes in thepicomolar to femtomolar (10⁻¹²-10⁻¹⁵ M) concentration range and lower.TABLE 1 BIOSENSOR COMBINATIONS Transducer Biological Element TypeExamples Electrochemical Enzymes Potentiometric Redox and ion-selectiveelectrodes (e.g., the Receptors pH electrode as well as CO₂, NH₃, andsulfide Micro-organisms electrodes based on this), FETS and LAPS Plantand animal tissues Enzyme-labeled antibodies Enzymes Amperometric Clarkoxygen electrode, mediated enzyme Micro-organisms electrodes Plant andanimal tissues Enzyme-labeled antibodies Enzymes Conductimetric Pt or Auelectrodes for determining the Bilayer lipid membranes* change inconduction of the solution due to the generation of ions OpticalReceptors Fluorescence Optrode, photodiodes, fiber-optic, bulk phaseAntibodies detection Enzymes Luminescence Optrode, photodiodes,fiber-optic, bulk phase detection Receptors Evanescent Coatedfiber-optic, fluorescence detection Antibodies wave Antibodies Surfaceplasmon resonance BIAcore (coated gold or silver layer on glass Antigenssupport), small haptens must be measured Enzymes indirectly bydisplacement assay Nucleic acids Antibodies Acoustic Piezoelectricdevices Antigens Enzymes Nucleic acids Enzymes Calorimetric Thermistoror thermopile Micro-organisms Cells*Man made.Enzymes

From an analytical point of view, the most important classes of enzymesare the oxidoreductases, which catalyse the oxidation of compounds usingoxygen or NAD, and the hydrolases, which catalyse the hydrolysis ofcompounds. Most successful biosensors exploit enzymes as the biologicalrecognition/response system because of the range of transduciblecomponents such as protons, ions, heat, light, electrons and mass thatcan be exchanged as part of their catalytic mechanism. This catalyticactivity is controlled by pH, ionic strength, temperature and thepresence of co-factors. Enzyme stability is usually the deciding factorin determining the lifetimes of enzyme based biosensors (typicallybetween 1 day and 1 or 2 months.

Organelles (e.g., mitochondria, chloroplasts) whole cells (e.g.,bacteria) or tissue sections from animal or plant sources have been usedas biocatalytic packages in biosensors for a large range of metabolitesof clinical interest. Together with the numerous enzymes present are allthe other necessary components needed to convert substrates intoproducts in an environment which has been optimized by evolution. Themajor drawback of the use of such systems is their multi-enzymebehavior, which results in decreased substrate specificity. However,sometimes such behavior can work to advantage because by merely changingthe external experimental conditions different substrates can bemeasured with the same biocatalytic material. The appropriate use ofenzyme inhibitors, activators and stabilizing agents also can be used toenhance the selectivity and lifetimes of tissue based biosensors.

Receptors

Naturally occurring receptors are non-catalytic proteins that span cellmembranes, extending into both the extracellular and intracellularspaces. They are involved in the chemical senses, such as olfaction andtaste, as well as in metabolic and neural biochemical pathways. Withinthe organism they act as links in cell-cell communication by reversiblybinding specific neurotransmitters and hormones liberated from othercells for the purpose of conveying messages through the target cell'smembrane to initiate or diminish its cellular activity. They are alsothe binding sites for many drugs and toxins. Two methods have beendefined by which binding of a transmitter molecule to the extracellularside of the receptor leads to modification of intracellular processes.

Attempts at using neuroreceptors as the recognition element inbiosensors have largely been restricted to the nicotinic acetylcholinereceptor (n-AChR) which can be isolated from the electric organ of theelectric eel or ray in relatively large quantities. The unavailabilityof other receptors for biosensor use is no doubt a reflection of thefact that they are normally only present in small amounts in tissues andare unstable once removed from their natural lipid membrane environment.However, the products of receptor DNA expression in foreign cell linesmay produce proteins useful for biosensor applications, yet not fullyidentical to the native starting material. The n-AChR and associated ionchannel complex binds several naturally occurring toxins.

Antigens and Antibodies

An antigen is any molecular species that can be recognized by an animalorganism as being foreign to itself and which therefore triggers thedefensive mechanism known as the immune response. This recognition has alower molecular weight cut-off of ˜10,000 Da. In natural circumstancessuch antigens are typically proteins or lipopolysaccharides at thesurfaces of viruses, bacteria and microfungi, or at the surfaces ofcells and in solution in blood or tissues of other species or even ofdifferent individuals of the same species. Foreign DNA or RNA is alsoantigenic as is material of plant origin.

An antibody (Ab) is a molecule produced by animals in response to theantigen and which binds to the latter specifically. Antibodies tosmaller molecular weight environmental contaminants such as pesticides,herbicides, microbial toxins and industrial chemicals can be made afterfirst covalently attaching the latter to a carrier protein such asbovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Thesmall molecular component of the resultant conjugate, which has beenmodified for antigenic recognition, is known as a hapten. A host ofother biotoxins of microbial, plant and animal origin are eitherantigenic or can be rendered antigenic by the formation ofhapten-protein conjugates.

In mammals, two distinct types of molecule are involved in therecognition of antigens. These are the proteins called immunoglobulinswhich are present in the serum and tissue fluids, and the antigenreceptors on the surface of specialized blood cells-the T-lymphocytes.It is the immunoglobulins, or antibodies, whose selective and tightbinding characteristics for antigens are made use of in immunologicalmethods of analysis. In most higher animals the immunoglobulins, orantibodies, fall into five distinct classes, namely IgG, IgA, IgM, IgDand IgE. These differ from each other in size, charge, amino acidcomposition and carbohydrate content. They all appear to beglycoproteins but the carbohydrate content ranges from 2-3% for IgG to12-14% for the others. The basic structure of all immunoglobulinmolecules is a Y-shaped unit consisting of two identical lightpolypeptide chains and two identical heavy polypeptide chains linkedtogether by disulfide bonds. The amino terminal ends of the ‘arms’ ofthe Y are characterized by sequence variability and are the antigenbinding sites. IgG is the exclusive anti-toxin class of antibody. IgM isa pentamer of five Y-shaped units whose role appears to be to complexinfectious organisms.

The binding of antigen to antibody at transducer surfaces can bemeasured directly and indirectly. Binding can be detected by conjugatingthe antigen or antibody to a fluorescent label.

Nucleic Acids

The specific sequence of bases along a strand of DNA and the uniquecomplementary nature of the pairing between the base pairs (adenine andthymine or cytosine and guanine) of adjacent strands in the double helixis the basis of biodiversity. The ability of a single-stranded nucleicacid molecule to recognize and bind (hybridize) to its complementarypartner in a sample has been used in genetic analyses and may also beused in a biosensor.

Sample preparation might include one or more of the following steps: (a)extraction of the DNA from the cells in a sample; (b) preparation of theDNA in single stranded form; and (c) increasing the total amount of DNApresent by the use of the polymerase chain reaction (PCR™).

Another possibility is to use DNA binding proteins such as RNApolymerases, promoters, repressors and restriction enzymes, whichexhibit the ability to bind to a specific DNA sequence in adouble-stranded form to develop a biosensor. Since the preparation ofthe DNA in single-stranded form and its subsequent hybridization wouldnot be required, the method would involve a shorter sample preparationtime.

Viruses, Bacteria and Fungi

Viruses are small cellular parasites that cannot reproduce bythemselves. They attach to cells via specific receptors and this partlydetermines which cell types become infected. The particular cells thatare infected are ultimately destroyed because of the complex biochemicaldisturbances accompanying the intracellular replication of the virus.Viruses contain either single-stranded or double-stranded RNA or DNA,which is generally surrounded by an outer shell of one or morevirus-specific proteins or glycoproteins. In some viruses there is afurther external envelope that consists mainly of lipids but alsocontains some virus-specific proteins. It is the surface coat proteins,which are the viral antigens that trigger the immune response andantibody production. Viruses (and bacteria) have a large number ofantigenic determinants on their surfaces and therefore each organism canbind a number of antibody units. This results in a considerable increasein stability of virus-antibody complexes over hapten-antibody complexes(up to 10³-10⁴-fold depending on the antibody). TABLE 2 PATHOGENICORGANISMS Viruses Bacteria Fungi Variola virus Rickettsia prowazeckiCoccidioides immitis Chikungunya virus Rickettsia rickettsi Histoplasmacapsulatum Eastern encephalitis virus Rickettsia tsutsugamushi Norcardiaasteroides Venezuelan encephalitis virus Bacillus anthracis Westernencephalitis virus Francisella (Pasteurellas tularensis) Dengue virusPasteurella pestis Yellow fever virus Brucella melitensis. B. suisJapanese encephalitis virus Coxiella burnetti Russian spring-summerencephalitis virus Salmonella typhi Argentine haemorrhagic fever virusSalmonella paratyphi Lassa fever virus Vibrio comma Lymphocytechoriomeningitis virus Corynebacterium diphtheria Bolivian haemorrhagicfever virus Actinobacillus mallei Crimean-Congo haemorrhagic fever virusPseudomonas pseudomallei Haantan (Korean haemorrhagic fever)Mycobacterium tuberculosis virus Rift Valley fever virus Marburg virusEbola virus Hepatitis A virus

Certain pathogenic bacteria synthesize and secrete exotoxins as part ofthe mechanism underlying the specific symptoms of the diseases that theyproduce. Examples of these proteins that poison or kill susceptiblemammalian cells are the Shigella dysenteria toxin. Staph. aureusenterotoxin, tetanus toxin and botulinum neurotoxin, as well as thetoxins produced by Bacillus anthracis and Corynebacterium diphtheriae.Other pathogenic bacteria (the Salmonella and Brucella species in Table2) liberate toxins when they are lysed. These toxins are components ofthe bacterial cell wall and are conjugates of protein, lipid andcarbohydrate and have been called endotoxins. Both types of toxin areantigenic. The different types of bacteria have different cell wallstructures. All types (Gram-positive (G+), gram-negative (G−) andmycobacteria) have an inner cell membrane and a peptidoglycan wall.Gram-negative bacteria also have an outer lipid bilayer in whichlipopolysaccharide is sometimes found. The outer surface of thebacterium may also contain fimbriae or flagellae, or be covered by aprotective capsule. Proteins and polysaccharides in these structures canact as targets for the antibody response.

Some fungi are pathogenic to man because they can invade the bodytissues and proliferate there rather than because they liberate toxins.Three of these are listed in Table 2. Other fungi are dangerous tohumans because of the toxins they produce and liberate into theenvironment. A particular example of the latter is the fusarium species,which produce tricothecene mycotoxins mentioned.

Fungi may be utilized as a bioreporter. The inventors contemplate theuse of fungi (e.g., yeast) in methods of the present invention. Forexample, yeast strains may be constructed by methods similar to thosedisclosed herein for bacterial strains in which the yeast will emit abioluminescent signal in response to an environmental signal or stress.

Biosensors Based on Antibodies

There is a wide range of toxins for which enzyme based and receptorbased strategies are not available for the development of biosensors.However, assuming that one can obtain the appropriate antibodies,antibody based biosensors are possible for several of toxic chemicalsand probably all toxins and pathogenic micro-organisms listed in Table2.

One of the continuing challenges in the development of immunosensors isto be able to immobilize the antibodies at high density on theappropriate surface whilst still maintaining their functionalconfiguration and preventing stearic hindrance of the binding sites.This has led to the use of self-assembling long chain alkyl membranesystems (SAMSs) on glass or silica and gold surfaces. The terminalfunctional groups on each chain are designed to react with specificgroups on antibodies or antibody fractions to form a uniform geometricalarray of antigen binding sites.

The stability of the immobilized antibodies is also a critical factorfor future immunosensor research. A problem associated with this is thatif on-site preparation of the system for the capture process isrequired, this may take several h and methods need to be developed tospeed this up. A further requirement which is more important forimmobilization on piezoelectric devices is the need to reducenon-specific protein binding to the sensor surface. Perhaps one approachto this problem would be to use a SAM formed from a mixture of two longchain alkane thiolates, one with a terminal functional group forreaction with, for example, Fab-SH groups and the other presenting ashort oligomer of ethylene glycol to resist the non-specific adsorptionof protein at the membrane surface (Mrksich & whitesides, 1995). Thismixture would allow the possibility of controlling the spacing of thecovalently bound antibody fraction and optimizing specific antigenbinding.

Most immunological reactions are essentially irreversible because oftheir large association constants (K_(a)s of 10⁵-10⁹ M⁻¹). The K_(a)Sare composed of large forward [k₁] and small reverse [k⁻¹] rateconstants ranging from 10⁷ to 10⁹ M⁻¹ s⁻¹ and 10² to 10⁻⁴ s⁻¹,respectively. Developing antibodies with sufficiently fast antigendissociation rates to allow reversible measurements in real time couldlead to continuous or at least sequential measurements of the antigenwithout the need to replace the antibody or reverse the binding by theuse of chaotropic solutions. Recombinant technology will eventuallyallow the production of antibodies with new binding properties.

An approach that may solve the problem of irreversibility is thedevelopment of catalytic antibodies. Haptens designed to mimic thestereoelectronic features of transition states can induce antibodiescapable of catalysing a wide range of chemical transformations, rangingfrom simple hydrolyses of esters and amides to reactions that lackphysiological counterparts or are normally disfavored. Thus, it isconceivable that if catalytic antibodies can be obtained for toxicchemicals and toxins, then biosensors for these substances, capable ofcontinuous unattended running and not requiring fresh supplies of sensormaterial, could become a reality.

Alternatively, to utilize immunoreactions effectively in sensor design,the problem of irreversibility may be circumvented by creating areservoir that passively releases immunoreagents to the sensing regionof the particular device. Controlled release polymers have been used forthis purpose.

Recently, Wallace and co-workers (see Sadik et al., 1994) have suggestedthat because the Ag-Ab interaction is a multi-step process (involving avariety of different molecular interactions according to the distanceapart), it is possible that specificity is locked in at the early stagesand irreversibility occurs at the later stages, accompanied byconformational changes. Wallace et al. have presented evidence of thisspecificity from pulsed amperometric measurements using a platinumelectrode coated in a film of polypyrrole containing the antibody tothaumatin. During continuous pulsing of the applied potential in thepresence of the antigen, rapid and reversible peaks of current wereobserved whose height was directly proportional to the antigenconcentration. Injections of BSA and other proteins gave very muchreduced responses but it is not clear how much of this was due to thedifference in charge structure.

Nucleic Acid Based Biosensors

The time consuming preparative steps in gene probe assays make itdifficult for them to be considered as the basis of biosensors for theon-site detection of pathogenic micro-organisms. The majortime-consuming steps are the DNA isolation and amplification (PCR™)procedures and the hybridization detection step. Recently, it has beenpossible to grow ss-DNA on the surface of optical fibers and to detectthe hybridization process with complementary ss-DNA in a sample by usingthe fluorescence of ethidium bromide trapped in the double-strandedregions of the bound DNA. Besides, the possibility of being verysensitive and selective, such a nucleic acid based sensor has someadvantages over antibody based biosensors. First, it is more stable andcan be stored for longer periods. Also, the probe can be repeatedlyregenerated for further use by a short immersion in hot buffer. Futurework will be directed towards developing appropriate DNA probes forpathogenic bacteria and fungi and improving the methods for immobilizingthem on the sensor surface. Detection of hybridization may be furtherimproved by covalently immobilizing the ds-DNA sensitive fluorescent dyedirectly onto the immobilized ss-DNA at the glass fiber surface.

Microbial Biosensors Using Multiplexed Bioluminescence

Whole cell biosensors are occasionally limited in terms of sensitivityand reliability by signal transduction mechanisms and by non-specificinterferences. Wood and Gruber (1996) provide an overview of accuratelytransducing the genetic sensing mechanisms of microbes into readilymeasurable signals. Because living cells are a steady-stage ensemble ofhundreds of interacting biochemical pathways, it is difficult to changeone path without affecting, to some degree, several other paths. Withmany potential internal and external conditions being sensedsimultaneously in a cell, a change in any one could affect the cellularphysiology in unpredictable ways. In a microbial sensor, this may leadto unreliable and even uninterpretable responses to environmentalconditions.

In order to make microbial sensors more reliable, it may be necessary toascertain the effects of the non-specific stimuli and eliminate themfrom the sensor response. The cumulative effect of the non-specificstimuli may be determined through a second signal transducer (notcoupled to the specific genetic sensing system) to yield an internalcontrol signal. This control signal may serve as a dynamic baseline withwhich to compare the target signal. Since the signal from the targettransducer indicates the effects of both the targeted and non-specificstimuli, by normalizing the target signal to the control signal, theeffects of the target stimulus can be isolated.

This may be achieved by employing two genetic reporters which behaveessentially identically within the complex chemical environment ofliving cells, but yield readily differentiated signals. In preferredembodiments, the two genetic reporters may include bioluminescenceproteins with subtle modifications between each other, suchmodifications providing a distinguishable change in emission wavelength.Examples of such variants are commercially available for manybioluminescent reporters and are well known in the art (see e.g., Wood,1990).

Multiple bioluminescent reporters may be constructed to be translatedinto a single polypeptide comprising two functional bioreporters. If theexcitation spectrum of one or more of the reporters is within the rangeof emission of one or more of the other reporters in such a construct,the inventors contemplate that this “hybrid” construct would provideincreased signal strength or sensitivity or both over those comprisingonly one reporter. Alternatively, as opposed to being translated into asingle polypeptide, the multiple bioluminescent reporters may betranslated into separate polypeptides that encode regions that allow thereporters to bind each other or be in close proximity to each other.“Close proximity” refers to an arrangement where the emission of one ormore of the reporters is able to excite one or more of the otherreporters.

Recombinant Vectors Expressing Bioluminescence Genes

One important embodiment of the invention is a recombinant vector whichcomprises one or more nucleic acid segments encoding one or morebioluminescence polypeptides. Such a vector may be transferred to andreplicated in a prokaryotic or eukaryotic host, with bacterial cellsbeing particularly preferred as prokaryotic hosts, and yeast cells beingparticularly preferred as eukaryotic hosts.

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes or promoters isolated from anybacterial, viral, eukaryotic, or plant cell, or both. Naturally, it willbe important to employ a promoter that effectively directs theexpression of the DNA segment in the cell type, organism, or evenanimal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology. The promoters employed may beconstitutive, or inducible, and can be used under the appropriateconditions to direct high level expression of the introduced DNAsegment.

In a first embodiment, the recombinant vector comprises a nucleic acidsegment encoding one or more bioluminescence polypeptides. Highlypreferred nucleic acid segments are the lux genes of Vibrio fischerii,luxCDABE. Other preferred nucleic acid segments may include, but are notlimited to, those that encode firefly luciferase, the luciferaseproteins of other beetles, Dinoflagellates (Gonylaulax; Pyrocystis),Annelids (Dipocardia), Molluscs (Lativa), Crustacea (Vargula;Cypridina), green fluorescent protein of Aequorea victoria or Renillareniformis, or luciferases from other organisms capable ofbioluminescence.

In a second embodiment of the present invention, the inventorscontemplate a recombinant vector comprising a nucleic acid segmentencoding one or more enzymes that are capable of producing a reactionthat yields a luminescent product or a product that can be directlyconverted to a luminescent signal. For example, substrates of thecommonly used □-galactosidase and alkaline phosphatase enzymes arecommercially available that are luminescent (chemiluminescence) whenconverted by the respective enzyme.

In a third embodiment of the present invention, the inventorscontemplate a recombinant vector comprising a nucleic acid segmentencoding one or more enzymes that are capable of producing a reactionthat yields a chromogenic product or a product that can be directlyconverted to a chromogenic signal. For example, substrates of thecommonly used □-galactosidase and alkaline phosphatase enzymes arecommercially available that are chromogenic when converted by therespective enzyme. Appropriate choices of excitation and emissionwavelengths will permit detection and quantization of the chromogeniccompound. Likewise, any chromogenic substrate for which a standard assayis available for spectrophotometric analysis should be readily adaptablefor use in the present methods.

In a fourth embodiment of the present invention, the inventorscontemplate a recombinant vector comprising a nucleic acid segmentencoding one or more polypeptides that expressed on the surface of acell, or secreted from a cell. In a preferred embodiment, the nucleicacid segment encodes one or more TnPhoA polypeptides. In anotherembodiment, the polypeptide is an antigen of an antibody that, directlyor indirectly, is capable of producing a bioluminescent,chemiluminescent, or chromogenic product.

In each of the above embodiments, the recombinant vector may comprisethe gene of interest operatively linked to a promoter that is responsiveto an environmental factor. In a preferred embodiment the lax genes ofVibrio fischerii, luxCDABE are operatively linked to the tod operonwithin a mini-Tn5 transposon. However, the inventors contemplate thatvirtually any recombinant vector that allows the nucleic acid segment ofinterest to be operatively linked to a promoter that is responsive to anenvironmental factor may be used. Useful recombinant vectors mayinclude, but are not limited to, the gene of interest operatively linkedto a promoter that is responsive to an environmental factor by means ofgene fusions, operon fusions, or protein fusions.

Another important embodiment of the invention is a transformed host cellwhich expresses one or more of these recombinant vectors. The host cellmay be either prokaryotic or eukaryotic, and particularly preferred hostcells are those which express the nucleic acid segment or segmentscomprising the recombinant vector which encode the lux genes of Vibriofischerii, luxCDABE. Bacterial cells are particularly preferred asprokaryotic hosts, and yeast cells are particularly preferred aseukaryotic hosts

A wide variety of ways are available for introducing a nucleic acidsegment expressing a polypeptide able to provide bioluminescence orchemiluminescence into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the nucleic acid segment, thenucleic acid segment under their regulatory control and a DNA sequencehomologous with a sequence in the host organism, whereby integrationwill occur or a replication system which is functional in the host,whereby integration or stable maintenance will occur or both.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In preferred instances, it may bedesirable to provide for regulative expression of the nucleic acidsegment able to provide bioluminescence or chemiluminescence, whereexpression of the nucleic acid segment will only occur after releaseinto the proper environment. This can be achieved with operators or aregion binding to an activator or enhancers, which are capable ofinduction upon a change in the physical or chemical environment of themicroorganisms. For translational initiation, a ribosomal binding siteand an initiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger RNA, particularly by using an active promoter, as well asby employing sequences, which enhance the stability of the messengerRNA. The transcriptional and translational termination region willinvolve stop codon or codons, a terminator region, and optionally, apolyadenylation signal (when used in an Eukaryotic system).

In the direction of transcription, namely in the 5′ to 3′ direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5′ or 3′ of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop codon orcodons, the polyadenylation signal sequence, if any, and the terminatorregion. This sequence as a double strand may be used by itself fortransformation of a microorganism host, but will usually be includedwith a DNA sequence involving a marker, where the second DNA sequencemay be joined to the expression construct during introduction of the DNAinto the host.

By “marker” the inventors refer to a structural gene which provides forselection of those hosts which have been modified or transformed. Themarker will normally provide for selective advantage, for example,providing for biocide resistance (e.g., resistance to antibiotics orheavy metals); complementation, so as to provide prototrophy to anauxotrophic host and the like. One or more markers may be employed inthe development of the constructs, as well as for modifying the host.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 basepairs (bp), preferably atleast about 100 bp, more preferably at least about 1000 bp, and usuallynot more than about 2000 bp of a sequence homologous with a sequence inthe host. In this way, the probability of legitimate recombination isenhanced, so that the gene will be integrated into the host and stablymaintained by the host. Desirably, the nucleic acid segment able toprovide bioluminescence or chemiluminescence will be in close proximityto the gene providing for complementation as well as the gene providingfor the competitive advantage. Therefore, in the event that the nucleicacid segment able to provide bioluminescence or chemiluminescence islost, the resulting organism will be likely to also have lost thecomplementing gene, and the gene providing for the competitiveadvantage, or both.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the λ_(L) and λ_(R) promoters, the tac promoter. See forexample, U.S. Pat. No. 4,356,270. The termination region may be thetermination region normally associated with the transcriptionalinitiation region or a different transcriptional initiation region, solong as the two regions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pR01614, and the like. See for example, U.S. Pat. No. 5,441,884,incorporated specifically herein by reference.

The desired gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against unmodified organisms or transferringorganisms, when present. The transformants then can be tested forbioluminescence or chemiluminescence activity. If desired, unwanted orancillary DNA sequences may be selectively removed from the recombinantbacterium by employing site-specific recombination systems, such asthose described in U.S. Pat. No. 5,441,884, specifically incorporatedherein by reference.

Methods for Preparing Antibodies

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a polypeptide. Reference to antibodies throughoutthe specification includes whole polyclonal and monoclonal antibodies(mAbs), and parts thereof, either alone or conjugated with othermoieties. Antibody parts include Fab and F(ab)₂ fragments and singlechain antibodies. The antibodies may be made in vivo in suitablelaboratory animals or in vitro using recombinant DNA techniques. In apreferred embodiment, an antibody is a polyclonal antibody.

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster or a guinea pig. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

Antibodies, both polyclonal and monoclonal, specific for givenpolypeptides may be prepared using conventional immunization techniques,as will be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of particular polypeptides can be used toimmunize one or more experimental animals, such as a rabbit or mouse,which will then proceed to produce specific antibodies against thepolypeptide. Polyclonal antisera may be obtained, after allowing timefor antibody generation, simply by bleeding the animal and preparingserum samples from the whole blood.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen, as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster injection, alsomay be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored or the animal can be used to generate mAbs (below), or both.

One of the important features provided by the present invention is apolyclonal sera that is relatively homogenous with respect to thespecificity of the antibodies therein. Typically, polyclonal antisera isderived from a variety of different “clones,” i.e. B-cells of differentlineage. mAbs, by contrast, are defined as coming fromantibody-producing cells with a common B-cell ancestor, hence their“mono” clonality.

When peptides are used as antigens to raise polyclonal sera, one wouldexpect considerably less variation in the clonal nature of the sera thanif a whole antigen were employed. Unfortunately, if incomplete fragmentsof an epitope are presented, the peptide may very well assume multiple(and probably non-native) conformations. As a result, even shortpeptides can produce polyclonal antisera with relatively pluralspecificities and, unfortunately, an antisera that does not react orreacts poorly with the native molecule.

Polyclonal antisera according to present invention is produced againstpeptides that are predicted to comprise whole, intact epitopes. It isbelieved that these epitopes are therefore more stable in an immunologicsense and thus express a more consistent immunologic target for theimmune system. Under this model, the number of potential B-cell clonesthat will respond to this peptide is considerably smaller and, hence,the homogeneity of the resulting sera will be higher. In variousembodiments, the present invention provides for polyclonal antiserawhere the clonality, i.e., the percentage of clone reacting with thesame molecular determinant, is at least 80%. Even higher clonality up to90% or 95% or greater is contemplated.

To obtain mAbs, one would also initially immunize an experimentalanimal, often preferably a mouse, with a polypeptide-containingcomposition. After a period of time sufficient to allow antibodygeneration, one would obtain a population of spleen or lymph cells fromthe animal. The spleen or lymph cells can then be fused with cell lines,such as human or mouse myeloma strains, to produce antibody-secretinghybridomas. These hybridomas may be isolated to obtain individual cloneswhich can then be screened for production of antibody to the desiredpolypeptide.

Following immunization, spleen cells are removed and fused, using astandard fusion protocol with plasmacytoma cells to produce hybridomassecreting mAbs against a polypeptide of interest. Hybridomas whichproduce mAbs to the selected antigens are identified using standardtechniques, such as ELISA and Western blot methods. Hybridoma clones canthen be cultured in liquid media and the culture supernatants purifiedto provide the polypeptide of interest-specific mAbs.

Of particular utility to the present invention are antibodies taggedwith a fluorescent or enzymatic molecule. Methods of tagging antibodiesare well known to those of skill in the art and a large number of suchantibodies are available commercially. Fluorescent tags include, but arenot limited to, fluorescein, phycoerythrin, and Texas red. Enzymatictags, include, but are not limited to, alkaline phosphatase andhorseradish peroxidase.

Nucleic-Acid Segments

The present invention also concerns nucleic acid segments that can beisolated from virtually any source, that are free from total genomic DNAand that encode bioluminescence peptides disclosed herein. Nucleic acidsegments encoding these peptide species may prove to encode proteins,polypeptides, subunits, functional domains, and the like of lux-relatedor other non-related gene products. In addition these nucleic acidsegments may be synthesized entirely in vitro using methods that arewell-known to those of skill in the art.

As used herein, the term “nucleic acid segment” refers to a nucleic acidmolecule that has been isolated free of total genomic nucleic acid of aparticular species. Therefore, a nucleic acid segment encoding abioluminescence peptide refers to a nucleic acid segment that contains abioluminescence polypeptide coding sequences yet is isolated away from,or purified to be free from, total genomic nucleic acid of the speciesfrom which the nucleic acid segment is obtained. Included within theterm “nucleic acid segment,” are nucleic acid segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a nucleic acid segment comprising an isolated or purifiedbioluminescence gene refers to a nucleic acid segment which may include,in addition to peptide encoding sequences, certain other elements suchas, regulatory sequences, isolated substantially away from othernaturally occurring genes or protein-encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein-, polypeptide- or peptide-encoding unit. As will be understoodby those skilled in the art, this functional term includes both genomicsequences, cDNA sequences and smaller engineered gene segments thatexpress, or may be adapted to express proteins, polypeptides orpeptides. In a preferred embodiment, the nucleic acid segment comprisesan operon of lux genes.

“Isolated substantially away from other coding sequences” means that thegene, or operon, of interest, in this case, an operon encodingbioluminescence polypeptides, forms the significant part of the codingregion of the DNA segment, and that the DNA segment does not containlarge portions of naturally-occurring coding DNA, such as largechromosomal fragments or other functional genes or cDNA coding regions.Of course, this refers to the DNA segment as originally isolated, anddoes not exclude genes or coding regions later added to the segment bythe hand of man.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

The various probes and primers designed around the disclosed nucleotidesequences of the present invention may be of any length. By assigningnumeric values to a sequence, for example, the first residue is 1, thesecond residue is 2, etc., an algorithm defining all primers can beproposed:n to n+ywhere n is an integer from 1 to the last number of the sequence and y isthe length of the primer minus one, where n+y does not exceed the lastnumber of the sequence. Thus, for a 10-mer, the probes correspond tobases 1 to 10, 2 to 11, 3 to 12, and so on. For a 15-mer, the probescorrespond to bases 1 to 15, 2 to 16, 3 to 17, and so on. For a 20-mer,the probes correspond to bases 1 to 20, 2 to 21, 3 to 22, and so on.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention. Recombinant vectors and isolated DNA segments may thereforevariously include the peptide-coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includethese peptide-coding regions or may encode biologically functionalequivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional equivalent peptides. Such sequences may arise asa consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by humans may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the bioluminescence of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asplastid targeting signals or “tags” for purification or immunodetectionpurposes (e.g., proteins that may be purified by affinity chromatographyand enzyme label coding regions, respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning or PCR™ technology, or both in connection with thecompositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a nucleic segment encoding one ormore bioluminescence polypeptides in its natural environment. Suchpromoters may include promoters normally associated with other genes, orpromoters isolated from any bacterial, viral, eukaryotic, or plant cell,or both. Naturally, it will be important to employ a promoter thateffectively directs the expression of the DNA segment in the cell type,organism, or even animal, chosen for expression. The use of promoter andcell type combinations for protein expression is generally known tothose of skill in the art of molecular biology, for example, seeSambrook et al., 1989. The promoters employed may be constitutive, orinducible, and can be used under the appropriate conditions to directhigh-level expression of the introduced DNA segment, such as isadvantageous in the large-scale production of recombinant proteins orpeptides. Preferred promoters are those that are induced in the presenceof environmental factors or stress.

The ability of such nucleic acid probes to specifically hybridize tobioluminescence polypeptide-encoding sequences will enable them to be ofuse in detecting the presence of complementary sequences in a givensample. However, other uses are envisioned, including the use of thesequence information for the preparation of mutant species primers, orprimers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of such as 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, etc.; 60, 61, 62, 63,etc.; 70, 71, 72, 73, etc., 80, 81, 82, 83, etc., 90, 91, 92, 93, etc.;100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including allintegers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000;3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about12,001, 12,002, 13,001, 13,002 and the like nucleotides or so, identicalor complementary to nucleic acid sequences disclosed herein areparticularly contemplated as hybridization probes for use in, e.g.,Southern and Northern blotting. Smaller fragments will generally finduse in hybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 10 to 14 andabout 100 or 200 nucleotides, but larger contiguous complementarystretches may be used, according to the length complementary sequencesone wishes to detect.

The use of a hybridization probe of about 14, 15, 16, 17, 18, or 19nucleotides in length allows the formation of a duplex molecule that isboth stable and selective. In order to increase stability andselectivity of the hybrid molecules having contiguous complementarysequences over stretches greater than 14, 15, 16, 17, 18, or 19 bases inlength are generally preferred and thereby improve the quality anddegree of specific hybrid molecules obtained; however, one willgenerally prefer to design nucleic acid molecules havinggene-complementary stretches of 15 to 20 contiguous nucleotides, or evenlonger, where desired.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202,incorporated herein by reference, by introducing selected sequences intorecombinant vectors for recombinant production, and by other recombinantDNA techniques generally known to those of skill in the art of molecularbiology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt, or high temperature conditions, such asprovided by about 0.02 M to about 0.15 M NaCl at temperatures of about50° C. to about 70° C., or both. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating bioluminescencepolypeptide-encoding DNA segments. Detection of DNA segments viahybridization is well known to those of skill in the art.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate bioluminescencepolypeptide-encoding sequences from related species, functionalequivalents, or the like, less stringent hybridization conditions willtypically be needed in order to allow formation of the heteroduplex. Inthese circumstances, one may desire to employ conditions such as about0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. toabout 55° C. Cross-hybridizing species can thereby be readily identifiedas positively hybridizing signals with respect to controlhybridizations. In any case, it is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature does. Thus, hybridization conditions canbe readily manipulated, and thus will generally be a method of choicedepending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescentand enzymatic, which are capable of giving a detectable signal. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as urease, alkaline phosphatase orperoxidase. In the case of enzyme tags, colorimetric indicatorsubstrates are known that can be employed to provide a means visible tothe human eye or spectrophotometrically to identify specifichybridization with complementary nucleic acid-containing samples.Similarly, in the case of fluorescent tags, fluorescent indicators areknown that can be employed to provide a means visible to the apparatusof the present invention.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.). Afterwashing of the hybridized surface so as to remove nonspecifically boundprobe molecules, specific hybridization is detected, or evenquantitated, by means of the label. Means for probe labeling and hybriddetection are well known to those of skill in the art.

Methods for Preparing Mutagenized DNA Segments

In certain circumstances, it may be desirable to modify or alter one ormore nucleotides in one or more of the promoter sequences disclosedherein for the purpose of altering or changing the transcriptionalactivity or other property of the promoter region. In general, the meansand methods for mutagenizing a DNA segment are well known to those ofskill in the art. Modifications to such segments may be made by randomor site-specific mutagenesis procedures. The promoter region may bemodified by altering its structure through the addition or deletion ofone or more nucleotides from the sequence which encodes thecorresponding unmodified promoter region.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art such as and not limited to synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular promoter region. In particular, site-specific mutagenesis isa technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector which includes within itssequence a DNA sequence which encodes the desired promoter region orpeptide. An oligonucleotide primer bearing the desired mutated sequenceis prepared, generally synthetically. This primer is then annealed withthe single-stranded vector, and subjected to DNA polymerizing enzymessuch as E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform or transfect appropriate cells, such as E. colicells, and clones are selected which include recombinant vectors bearingthe mutated sequence arrangement. A genetic selection scheme was devisedby Kunkel et al. (1987) to enrich for clones incorporating the mutagenicoligonucleotide. Alternatively, the use of PCR™ with commerciallyavailable thermostable enzymes such as Taq polymerase may be used toincorporate a mutagenic oligonucleotide primer into an amplified DNAfragment that can then be cloned into an appropriate cloning orexpression vector. A PCR™ employing a thermostable ligase in addition toa thermostable polymerase may also be used to incorporate aphosphorylated mutagenic oligonucleotide into an amplified DNA fragmentthat may then be cloned into an appropriate cloning or expressionvector.

The preparation of sequence variants of the selected promoter-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants of DNA sequences may beobtained. For example, recombinant vectors encoding the desired promotersequence may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing. Typically, vector mediated methodologies involve theintroduction of the nucleic acid fragment into a DNA or RNA vector, theclonal amplification of the vector, and the recovery of the amplifiednucleic acid fragment.

A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample. One of the best-knownamplification methods is the polymerase chain reaction (PCR™). Briefly,in PCR™, two primer sequences are prepared which are complementary toregions on opposite complementary strands of the target sequence. Anexcess of deoxynucleoside triphosphates are added to a reaction mixturealong with a DNA polymerase (e.g., Taq polymerase). If the targetsequence is present in a sample, the primers will bind to the target andthe polymerase will cause the primers to be extended along the targetsequence by adding on nucleotides. By raising and lowering thetemperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products and excess primerswill bind to the target and to the reaction products. The process isthen repeated. Preferably a reverse transcriptase PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Another method for amplification is the ligase chain reaction (referredto as LCR). In LCR, two complementary probe pairs are prepared, and inthe presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs.

Q-beta Replicase may also be used as still another amplification methodin the present invention. In this method, a replicative sequence of RNAwhich has a region complementary to that of a target is added to asample in the presence of an RNA polymerase. The polymerase will copythe replicative sequence which can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e. nick translation. Asimilar method, called Repair Chain Reaction (RCR), is another method ofamplification which may be useful in the present invention and isinvolves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. A similar approach is used in SDA.

Still other amplification methods may be used in accordance with thepresent invention. In one application, “modified” primers are used in aPCR™ like, template and enzyme dependent synthesis. The primers may bemodified by labeling with a capture moiety (e.g., biotin) and/or adetector moiety (e.g., enzyme). In another application, an excess oflabeled probes are added to a sample. In the presence of the targetsequence, the probe binds and is cleaved catalytically. After cleavage,the target sequence is released intact to be bound by excess probe.Cleavage of the labeled probe signals the presence of the targetsequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS) include nucleic acid sequence basedamplification (NASBA) and 3SR. In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a sample, treatment with lysis buffer and minispincolumns for isolation of DNA and RNA or guanidinium chloride extractionof RNA. These amplification techniques involve annealing a primer whichhas crystal protein-specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second crystalprotein-specific primer, followed by polymerization. The double strandedDNA molecules are then multiply transcribed by a polymerase such as T7or SP6. In an isothermal cyclic reaction, the RNAs are reversetranscribed into double stranded DNA, and transcribed once against witha polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate crystal protein-specific sequences.

A nucleic acid amplification process involving cyclically synthesizingsingle-stranded RNA (“ssRNA”), single-stranded DNA (ssDNA), anddouble-stranded DNA (dsDNA), may be used in accordance with the presentinvention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H(RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein byreference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target ssDNA followed by transcription of many RNA copiesof the sequence. This scheme is not cyclic, i.e. new templates are notproduced from the resultant RNA transcripts. Other amplification methodsinclude “RACE”, and “one-sided PCR™” which are well known to those ofskill in the art.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification of DNA sequences of the presentinvention.

Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments, which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons listed in Table 3. TABLE 3 TABLE OF CODONS Amino Acids CodonsAlanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp DGAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU GlycineGly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUCAUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as antigen-binding regions ofantibodies or binding sites on substrate molecules. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence; and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Bioluminescent Bioreporters

In prokaryotes, a bioluminescent bioreporter, designated lux, consistsof a luciferase composed of two different subunits coded by the genesluxA and luxB that oxidize a long chain fatty aldehyde to thecorresponding fatty acid resulting in a blue-green light emission near490 nm (Tu and Mager, 1995). The system also contains a multienzymefatty acid reductase consisting of three proteins (a reductase encodedby luxC, a transferase encoded by luxD, and a synthetase encoded byluxE) which initially converts and recycles the fatty acid to thealdehyde substrate. Thus, no exogenous addition of substrate is requiredto induce luminescence. The genes are contained on a single continuousoperon. This genetic configuration allows the cloning of the completelux gene cassette (reporter genes) downstream from different promotersfor the utilization of bioluminescence to monitor gene expression.

Implicit in the use of a bioreporter strain for a BBIC is that thebioluminescent signal generated is directly related to the concentrationof the target substance, most desirably in a selective and quantitativemanner. In general, lux reporter genes are placed under the regulatorycontrol of inducible operons maintained in native plasmids, broad hostrange plasmids, or chromosomally integrated into the host strain. Inthese genetic systems, the target analyte or its degradation productsact as the inducer of the bioluminescence genes, and are responsible forselectivity and the resultant response. For example, P. fluorescens HK44is a bioreporter that produces light in the presence of naphthalene.This strain has two genetic operons positively regulated by a LysRprotein. One of the operons contains the lux bioluminescence genes andthe other the genes responsible for the degradation of naphthalene tosalicylate, the metabolic intermediate of naphthalene degradation. Bothoperons are induced when salicylate interacts with the regulatoryprotein NahR. Exposure of HK44 to either naphthalene or salicylateresults in increased naphthalene gene expression and increasedbioluminescence.

Studies in continuous cultures of P. fluorescens HK44 have demonstratedthat the magnitude of the bioluminescence response correlated with theaqueous phase concentration of naphthalene under dynamic pulsedperturbation conditions (King, 1990). Reproducible bioluminescence wasobserved not only in aqueous naphthalene samples but also in soil slurrysamples which were spiked with naphthalene, complex soil leachates, andthe water soluble components of jet fuel (Heitzer, et al., 1992). P.fluorescens HK44 can be applied in environmental use for eitherquantitative analysis of contaminant presence or bioavailability.However, for such applications both the chemical complexity of theenvironment and the physiological conditions of the organisms must beconsidered in interpreting the bioluminescence response.

Many types of bioluminescent (lux) transcriptional gene fusions havebeen used to develop light emitting bioreporter bacterial strains tosense the presence, bioavailability, and biodegradation of otherpollutants including toluene (Applegate, et al., 1997), andisopropylbenzene (Selifonova, et al., 1993) Analogous genetic approacheshave also been reported for inducible heavy metal detoxification andresistance systems including mercury as well as the heat shock responseand response to oxidative stress. In addition, genetically engineeredGram positive bioreporters have been used to examine the efficacy ofantimicrobial agents where decreased light was equated to greaterefficacy (Andrew and Roberts (1993). Eukaryotic bioreporters have alsobeen generated to detect toxic compounds (Andrew and Roberts, 1993),oxygen, ultraviolet light, and estrogenic and antiestrogenic compounds(Anderson, et al., 1995). Environmental applications involvingbioluminescence measurements have been reviewed (Steinberg and Poziomek,1995).

Cell Entrapment

Various methods exist for the entrapment of microbial cells at or nearthe light-sensing portion of the IC. For instance, cells can be simplyentrapped behind a porous membrane or encapsulated in natural orsynthetic polymers.

Polymeric matrices can provide a hydrated environment containingnutrients and co-factors needed for cellular activity and growth. Inaddition, encapsulated cells are protected from toxic substances intheir environment and maintain increased plasmid stability. Cells can beencapsulated in thin films or small diameter beads in order to beadaptable to the small surface area available on the IC. Thin films canbe formed by mixing cells in a liquid polymer that is then micropipettedon the IC in a thin layer and allowed to polymerize. Larger blocks ofcells can also be made from which films of virtually any desiredthickness can be sliced and attached to the IC. Microbeads are producedby spraying the liquid polymer/cell mixture through a nebulizer into apolymerizing agent.

Sol-gels have been used to exemplify a suitable encapsulation medium.Sol-gel is a silica-based glass that polymerizes under room temperatureconditions. Although sol-gel has been used to encapsulate yeast cells,the reaction conditions necessary for polymerization (primarily low pH)are generally too harsh for bacterial cell immobilizations. Utilizingsonication methods, polymerization under pH conditions conducive to cellsurvival has been achieved. Toluene bioreporter (Pseudomonas putidaTVA8) and a naphthalene bioreporter (Pseudomonas fluorescens HK44) havesuccessfully been encapsulated in sol-gel and shown to producebioluminescence when exposed to their specific inducers. However,cracking and drying within the thin sol gel matrices afterpolymerization may occur.

Alternatively, an alginate polymerization matrix may be utilized foron-chip applications. Alginate lacks the structural integrity ofencapsulation agents such as sol gels, but has a significant advantagebecause of its straightforward adaptability to microbial encapsulationand its subsequent widespread use in cellular immobilization procedures.To increase mechanical stability, alginate encapsulated cells have beenentrapped in 0.1 μm low adsorption/absorption filter membranes andhollow fiber membranes which allow for influx of chemical analytes whileinhibiting alginate degradation and cellular release into thesurrounding medium. Lyophilization is expected to increase long termstorage of the encapsulated cells.

Additional Aspects of the Present Invention

In addition to the embodiments described in detail herein, the inventorsfurther contemplate that the BBIC of the present invention may be usedto detect pollutants, explosives, heavy-metals, or other chemical orbiological agents residing in areas like groundwater, streams, rivers,oceans, or other environments. Furthermore, the BBIC of the presentinvention may be used in combinatorial chemistry in biomedical-drug andanti-cancer screening, sensors for oil exploration, industrial processcontrol, and biomedical instrumentation. The BBIC of the presentinvention may be used to respond to the absence or low abundance of testchemicals, e.g., Fe⁺² or PO₄ ⁻³. In addition to compounds, the BBIC ofthe present invention may be used to detect environmental conditions,such as temperature, radiation, and pressure. The inventors contemplatethat essentially any signal transduction pathway may be utilizedprovided the organism of the BBIC is capable of detecting the presenceor absence of a substance or condition and alter the expression of apromoter operatively linked to a reporter gene.

Besides those described in detail herein, the inventors contemplateadditional methods of powering the bioluminescent bioreporter integratedcircuits (BBICs) of the present invention. They may be powered remotelyby induction of RF, optical energy (including solar), mechanical energy(vibration, water flow, air flow, etc.), chemical energy, or thermalenergy. In some embodiments of the present invention, the lightgenerated by the sensing organism or compound may be used to power theBBIC.

The inventors contemplate that the BBICs of the present invention may bereadout by wireless means (e.g., RF or on-chip light-emitting device) oralternatively, wired means (e.g., direct analog, digital, or passivemeans including resistance, capacitance, and inductance, etc.). TheBBICs of the present invention may also be realized in bipolar silicon,silicon-germanium, GaAs, InP or other semiconductor IC processes.

The light-emitting agent of the present invention may be biological orchemical, wherein the light producing mechanism may be luminescence,fluorescence, or phosphorescence. The inventors further contemplate thatthe light emitting agent may be placed on the IC at the time ofmanufacture or selected and placed on the IC at the time of use. Inother embodiments of the present invention, the inventors contemplateBBICs comprising arrays of light-emitting agents further comprising amatching array of light-detection devices. With the addition of signalprocessing (analog, digital, neural network, etc.), this array devicemay be used to detect a family of chemicals instead of an individualchemical. Additionally, the BBICs comprising arrays of light-emittingelements with different emission wavelengths further comprise anintegrated photo-spectrometer. For example, by measuring the spectra ofthe emitted light, this embodiment may be used to detect a number ofchemicals simultaneously or sequentially, instead of detecting a singlechemical.

A number of methods of packaging the BBICs of the present invention maybe envisioned. Generally, the type of packaging chosen may reflect thepredicted environment to which the BBIC would be subjected. Suchenvironments may include, but are not limited to, aqueous, gaseous, orsolid environments. For example, the inventors contemplate a BBICencased in concrete near a rebar to detect corrosion. In anotherembodiment, the inventors contemplate packaging the BBIC in a mannerthat may allow in vivo measurements for biomedical application (e.g.,detecting disease, sensing a patient's condition, etc). Generally, theBBIC would be packaged in a semi-permeable membrane that would permitthe particular fluid being examined (e.g., blood) to pass, whilesubstances which would harm or interfere with the BBIC (e.g., a animalhost defense mechanism) would be blocked.

In certain embodiments, the light-emitting agent of the presentinvention may comprise a multicellular organism (e.g., an insect). It iswell known that larger organisms such as insects can be geneticallyengineered to bioluminesce in the presence of targeted substances. Insuch cases, the IC portion of the BBIC may be attached to an insect insuch a way that the chip would detect the resulting bioluminesce. Such asystem would be mobile, since the insect itself is mobile and unaffectedby the presence of the attached BBIC. One such example of thisapplication of the apparatus disclosed herein is illustrated in FIG. 33.The inventors further contemplate that when the light-emitting agent isa multicellular organism, the BBIC may be self-propelling and/orself-powering.

The inventors contemplate a BBIC comprising global position sensing thatmay allow the BBIC to sense location as well as the presence or absenceof a certain compound or biological agent.

The inventors contemplate an array of BBICs connected in a wired orwireless distributed network to form an artificially intelligent sensingnetwork. This array of BBICs may comprise on-chip processing capabilityon each BBIC.

BBICs could be distributed over a wide area, yet wirelessly connectedtogether as shown in FIG. 34. If each BBIC had on-chip signal processingcapabilities (e.g., neural network processing), this distributed networkwould form an artificially intelligent sensor system. For example,consider a large network of BBICs deployed over a large area where atoxic gas leak has occurred. As the gas cloud enters the area of theBBIC network useful information such as gas composition, speed, anddirection of the cloud could be determined by the sensor network. Ifother information such as wind conditions, terrain topology,temperature, etc., were available to the network, the network could makepredictions of risks to human populations.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 A Modified Mini-Tn5 system for Chromosomally-Introduced luxReporters

This example describes a cloning plasmid which allows inserts to bedirectionally cloned into a mini-Tn5 transposon vector. Such vectors areuseful for preparing the bioreporter constructs useful in the methods ofthe present invention. As an exemplary embodiment, a tod-lux fusion wasconstructed and introduced into Pseudomonas putida F1 to examine theinduction of the tod operon when exposed to BTEX compounds and aqueoussolutions of JP-4 jet fuel constituents. Since this system contains thecomplete lux cassette (luxCDABE), bacterial bioluminescence can bemeasured in whole cells without the need to add an aldehyde substrate.The resultant strain was also evaluated for its stability and fitnesscompared to the wild type strain F 1.

Materials and Methods

Organisms and Culture Conditions

Strains used in these studies are shown in Table 4. All cultures weregrown at 28° C. except for E. coli strains, which were grown at 37° C.

DNA Isolation and Manipulation

Large scale plasmid DNA isolation was accomplished using a modifiedalkaline lysis protocol (Promega, 1992). Chromosomal DNA was preparedusing the protocol outlined by Ausubel et al. (1989). All DNApreparations were further purified by CsCl EtBr ultracentrifugation(Sambrook et al., 1989). DNA modifications and restriction endonucleasedigestions were performed.

Cloning and Transposon Construction

The transposon mini-Tn5KmNX was constructed using site-directedmutagenesis and the polymerase chain reaction. Two 58 baseoligonucleotides, 5′ and 3′ with respect to the kanamycin resistancegene (Km^(R)) in PCRII™ (Invitrogen, San Diego, Calif.) were synthesizedusing a Beckman Oligo 1000 DNA synthesizer (Palo Alto, Calif.) followingthe manufacturer's protocol. Base substitutions were made to generateboth I and O insertion sequences as well as unique NotI and XbaI sitesinside the transposon for cloning. An EcoRI site and a NheI site wereadded to the end of each oligonucleotide, respectively, to allow cloningof mini-Tn5 KmNX into the delivery vector pUT (Herrero et al., 1990).The sequence and base changes can be seen in FIG. 10. The primers wereused to amplify the kanamycin resistance gene from pCRII™ usingTouchdown PCR™ (Don et al., 1991) using the manufacturer's protocol withthe following thermocycler conditions: 94° C. initial denaturation, 5min; 5 cycles at 94° C. for 1 min, 72° C. annealing for 1 min, 72° C.extension for 2 min; the annealing temperature was lowered 5° C. every 5cycles until 42° C. at which 8 cycles were run, followed by a finalextension of 15 min at 72° C. The 1.3 kb product was cloned into pCRII™using a TA cloning kit (Invitrogen, San Diego, Calif.) according to themanufacturer's protocol. The resultant plasmid pUTK210 containing themini-Tn5KmNX was sequenced to verify the incorporation of both the 1 and0 insertion sequences. After confirmation, pUTK210 was cleaved with NheIand EcoRI and gel-purified using agarose gel electrophoresis. Thepurified mini-Tn5 KmNX fragment was cloned into the XbaI-EcoRI site ofthe mini-Tn5 delivery vector, pUT and electroporated into E. coli S 17-1(λpir). Electroporants with the proper inserts were selected on LBplates with 50 μg/ml kanamycin. DNA minipreps were obtained and insertswere verified by cleavage with restriction endonucleases.

The cloning vector, pLJS was constructed from pBluescript II (KS)(Stratagene, LaJolla Calif.) by cleaving with BssH II and religating toremove the multicloning site (MCS). Ligated DNA was transformed intoDH5a and spread on LB plates supplemented with ampicillin (50 μg/ml) andX-gal (40 μg/ml). Transformants without the MCS were white since theywere incapable of α-complementation. The resultant plasmid was namedpBSMCS(−). Two oligonucleotides (a 47-mer and a 44-mer) with basesubstitutions were synthesized as previously described to regenerate themulticloning site and add the following restriction sites, XbaI, NheI,SpeI, and AvrII. The sequences and orientation of the added sites can beseen in FIG. 10. The new multicloning site was amplified frompBluescript II (KS) using the manufacturer's protocol with the followingthermocycler conditions: 94° C. initial denaturation 5 min; 38 cycles ofdenaturation at 94° C. for 30 sec, annealing at 42° C. for 1 min,extension at 72° C. for 30 s; and, final extension at 72° C. for 15 min.The amplified fragment was cleaved with BssHII, ligated into pBSMCS(−)and transformed into DH5α™. Transformants were screened on LB agar withampicillin (50 μg/l) and X-gal (40 μg/ml). Blue colonies were selectedsince they indicated restored α-complementation. The construct wassequenced to confirm the base substitutions and integrity of the MCS.pLJST2 was generated by directionally-cloning the 0.77 kb HindIII-HincIIfragment containing the 5S ribosomal rrnB T₁T₂ transcription terminatorfrom pKK223-3 (Pharmacia, Piscataway, N.J.) into pLJS cleaved withHindIII and SmaI. The NotI-AvrII terminator fragment from pLJST2 wassubsequently cloned into the NotI-XbaI site of mini-Tn5KmNX. Thisallowed for the subsequent destruction of the XbaI site by heterologousligation and the regeneration of the NotI and XbaI unique sites inmini-Tn5KmNX downstream of the terminator (pUTK211). Mini-Tn5 Kmtod-lux(pUTK214) was generated by directionally cloning the 10.2 kb NotI-XbaItod-lux fragment from pUC 18 Not tod-lux (Table 4) into the NotI-XbaIsite of pUTK211. Both insert and vector DNA were purified by agarose gelelectrophoresis and electroelution before cloning. All other plasmidsand relevant constructs are described in Table 4. TABLE 4 PLASMIDSPlasmid Relevant Genotype/Characteristics pDTG514 pGem3Z with a 2.75 kbEcoRI-SmaI fragment from pDTG350 containing the tod promoter, P_(tod),Ap^(R) pUCD615 Promoterless luxCDABE cassette, ori pSa, ori pBR322,Ap^(R), Km^(R) pKK223-3 Expression vector containing the 5S ribosomalterminator rrnB T₁T₂ pBSKS pBluescript IIKS⁺ with multicloning site(MCS) KpnI-SacI, Ap^(R) pBSMCS(−) pBluescript without the MCS (BssHII-BssH II fragment removed), Ap^(R) pLJS pBSMCS(−) with added XbaI,NheI, AvrII and SpeI sites, Ap^(R) pLJS-tod pLJS containing the 1.8 kbSmaI-Xho I tod promoter fragment from pDTG514, Ap^(R) pLJS-lux pLJScontaining the 8.35 kb KpnI-PstI luxCDABE cassette from pUCD615, Ap^(R)pLJST2 pLJS containing the 0.77 kb HindIII-Hinc II fragment frompKK223-3 cloned into HindIII-SmaI site, Ap^(R) pUC18 Not Cloning vectorcontaining multicloning site flanked by NotI sites, Ap^(R) pUC18 Not-luxContains the 8.35 kb XbaI-Pst I fragment from pLJS-lux, Ap^(R) pUCI8Not- Contains the 1.8 kb SpeI-XhoI fragment from pLJS-tod, Ap^(R) todluxpUT 5.2 kb cloning vector containing mob RP4, ori R6K and Tn5 tnplacking NotI sites, Ap^(R) pCR ™ II 3.9 kb cloning vector for PCR ™products with 3′ A overhangs, Ap^(R), Km^(R) pUTK209 pCR ™ II containingmini-Tn5KmNX with unique NotI and XbaI sites, Ap^(R), Km^(R) pUTK210 pUTcontaining mini-Tn5KmNX, Ap^(R), Km^(R) pUTK211 pUT/mini-Tn5KmT2containing the 0.8 kb NotI-AvrII rrnB T₁T₂ fragment, Ap^(R), Km^(R)pUTK214 pUT/mini-Tn5Kmtod-lux containing the 10.2 kb NotI-XbaI fragmentfrom pUC18 Not-todlux, Ap^(R), Km^(R)

Electroporation

Electrocompetent cells were prepared as outlined by the manufacturer(BTX, San Diego, Calif.). Electroporations were performed using a BTXElectroporator 600 with the following conditions: 40 μl cells, 1 μlligation mixture, a 2.5 kV pulse for about 4.7 ms using a 2 mm gapcuvette. After the pulse, cells were immediately resuspended in LB (to 1ml) and allowed to recover for 1 h at 37° C. (200 rpm) before plating onLB plates with the appropriate antibiotic selection.

DNA Sequencing

The mini-Tn5 KmNX in pCRII™ was sequenced to confirm that thesite-directed mutagenesis was successful using both the forward andreverse sequencing primers for pCRII™. Sequencing was performed using anApplied Biosystems Model 373A (Foster City, Calif.).

Transposon Mutagenesis

E. coli S 17-1 (λpir) containing pUTK214 was mated into P. putida F1 byplate mating. Donor and recipient cells were mixed in a ratio ofapproximately 5 to 1, spotted onto LB plates and incubated at 25° C. for24 h. Mutants were selected on Pseudomonas isolation agar supplementedwith 50 μg/ml kanamycin. Colonies were subsequently sub-cultured to gridplates and exposed to toluene vapor. Colonies which produced light weregrown in mineral salts media (MSM) (Stanier et al., 1966) with toluenevapor to ascertain whether or not the transposon had inserted into arequired gene for the cell. The strains were also evaluated for theirperformance as bioreporters in liquid growing cell assays (Heitzer etal., 1992).

Confirmation of Transposition

The selected strain was subjected to DNA:DNA hybridization to verifytransposition as opposed to recombination by using a ³²P-labeled probespecific for the Tn5 transposase (tnp) contained on pUT. Equal targetamounts of luxA, todC and tnp DNA were loaded onto a Biotrans™ nylonmembrane (ICN, Irvine, Calif.) using a Bioslot blot apparatus (Biorad,Hercules, Calif.) according to the manufacturer's protocol. The blotconsisted of chromosomal DNA from F1, TVA8 and the aforementionedcontrols. The DNA was loaded in triplicate and the blot was subdividedand each separate blot was hybridized with either luxA, todC, or tnpPCR™-generated ³²P-labeled DNA probes. Blots were hybridized and washedas previously described (Applegate et al., 1997).

Stability Assays

Batch stability assays were performed by transferring 1 ml of a 100 mlovernight culture grown on LB with 50 μg/ml kanamycin (Km₅₀) to a 250 mlErlemeyer flask using toluene as a sole carbon source as described forthe growth curves. One ml of culture was transferred every day for fivedays to flasks with 100 ml MSM supplied with toluene vapor (without Km).Assays were performed in triplicate. Before each transfer, cells wereplated on selective (LBKm₅₀) and non-selective media (LB) to ascertainloss of kanamycin-resistance resulting from deletion or excision of thetransposon. Colonies were subjected to colony hybridization using a 295bp luxA DNA probe (Johnston, 1996).

Stability was also assayed in continuous culture using a New BrunswickBio Flow fermentor (Edison, N.J.) with a 370 ml vessel operated at 28°C. at 180 rpm. The feed consisted of MSM supplemented with toluene atapproximately 100 mg/L at a flow rate of 1.0 ml/min. This wasaccomplished by simultaneously adding toluene saturated-MSM at a flowrate of 0.2 ml a min and MSM at a flow rate of 0.8 ml a min using FMImetering pumps (Oyster Bay, N.Y.). The chemostat was maintained at 28°C. using a cold finger and a refrigerated circulating water bath(Brinkman, Westbury, N.Y.). The chemostat was operated for 14 days,which corresponded to about 100 generations. Monitoring for bothbioluminescence and optical density was performed daily. Cells from thechemostat were also plated every 7 days and colony hybridizations wereperformed as described previously.

Growth Curves

Growth curves of TVA8 and F1 were obtained by growing cells in 100 mlMSM with toluene vapor supplied as a sole carbon source in 250 mlErlemeyer flasks. Cultures were started from a fresh overnight culture,grown to an ODS₅₄₆ of 1.0 in 100 ml of LB and washed twice in 100 ml MSMand resuspended in 100 ml of media. A one ml aliquot of this suspensionwas added to the toluene flasks. The cultures were shaken at 200 rpm at28° C. and sampled approximately every hour. The OD₅₄₆ was measured foreach culture and rates of increase in optical density were determinedfrom the linear portion of the curves.

Bioluminescence Sensing

Bioluminescent assays were conducted as described by Heitzer et al.(1992). An overnight culture from a frozen stock of TVA8 was prepared ina 250 mL Erleumeyer flask containing 100 mL LB with 50 μg/ml kanamycin.A sub-culture was prepared in yeast extract-peptone-glucose media(YEPG), grown to an OD₅₄₆ of 0.35-0.45 and assayed every 30 min. Inpreliminary studies, an incubation time of 2 hours was shown to providea consistent light response which maximized the signal intensity. After2 hours, the final OD₅₄₆ was measured and values are expressed asspecific bioluminescence (namp/OD₅₄₆).

Test Sample Preparation

An aqueous solution of JP-4 jet fuel constituents was prepared by addingJP-4 to sterile deionized water in a 1 to 10 jet fuel to water ratio.The solution was shaken on a rotary shaker for 24 hours. After phaseseparation, aqueous phase aliquots were added to test vials. Testsolutions of toluene, benzene, ethylbenzene, phenol and isomers ofxylene were prepared as above.

Bioluminescence Measurements

Sample vials were placed in a light-tight box and the light output wasmeasured using a liquid light pipe and an Oriel photomultiplier anddigital display (Model 77340 and Model 7070, Stratford, Conn.), using 25mL scintillation vials were used. Bioluminescent readings were takenevery 30 minutes. Light measurements for growth curves and the chemostatwere measured as above with the exception that the light-tight box wasmodified to hold a cuvette allowing for light measurement after ODreadings.

Results

Strain Construction

Sequence analysis of the resultant mini-Tn5 KmNX showed that both the 1and 0 insertion sequences were identical to the primers that were usedto generate the transposon (see FIG. 10). The extra adenine that wasmistakenly added did not affect the construct. The plasmid pLJS (FIG.12) was also sequenced to confirm that the added sites were incorporatedand to determine the integrity of the multicloning site (MCS). Sequencedata showed that the MCS and all of the added sites were intact. Theresultant cloning vector also maintained the ability forα-complementation. A schematic representation of the mini-Tn5 KmNXconstruction and the final construct mini-Tn5 Kmtod-lux can be seen inFIG. 11.

The S 17-1 (λpir) strain of E. coli harboring mini-5Kmtod-lux was matedwith F1 and resultant mutants were screened for their ability to producebioluminescence when exposed to toluene. Fourteen strains were evaluatedfor their ability to grow on toluene MSM and number 8 was chosen anddesignated TVA8. The strain was examined to confirm that it was a resultof a transposition event and not a recombination event. DNA:DNAhybridization showed that TVA8 contained the lux genes but did not showhybridization with tnp. Blots hybridized with tnp were re-probed withtodC to verify that DNA was present. The negative transposase resultconfirmed that transposition had occurred.

Stability of TVA8

In stability studies with batch and continuous cultures, the transposoninsertion in F1 appeared to be stable. Plate counts from selective(LBKm₅₀) and non-selective media (LB) were compared to determine whetherthe kanamycin marker was being lost, and colony blots were subsequentlyhybridized with luxA probe to confirm that all colonies contained thelux transposon insert (and were not contaminants). For the succinatechemostat without antibiotic selection, the selective plate counts wereapproximately five percent lower than the non-selective plate countsafter 10 days, however, 100% of colonies from both plate types werelux-positive. In batch stability studies with toluene vapor supplied assole carbon source, TVA8 did not demonstrate instability when subjectedto the same evaluation. The selective:non-selective plate count ratiowas 1.12±0.13 after 5 daily transfers, and all colonies hybridized withthe luxA probe. Similar results were observed for TVA8 stability undercontinuous culture conditions with toluene supplied at approximately 100mg/L. After a 14 day period (approximately 100 generations), theselective:non-selective plate count ratio was 1.05±0.13 and all coloniesfrom selective and non-selective plates were lux-positive.

Quantitative response of tod-lux Reporter Strain to Toluene, BTEXCompounds and JP-4 Jet Fuel

An increase in bioluminescence response for increasing tolueneconcentrations was observed (see FIG. 13). The bioluminescence responseto toluene concentration over the range, 5 to 20 mg/L was linear withspecific bioluminescence values of 133 to 228 namp/OD₅₄₆. The foldincrease in light response for concentrations above 20 mg/L was less,showing 290 namp/OD₅₄₆ for 50 mg/L. The overall bioluminescence responsecurve showed a Michaelis-Menten (enzyme kinetics) shape, showingsaturation at higher inducer concentrations. The toluene detection limitwas determined to be less than 50 μg/l.

TVA8 was examined for its bioluminescence response to BTEX compounds aswell as phenol and water-soluble JP-4 jet fuel components. There was asignificant light response to benzene, m- and p-xylenes, phenol and JP-4(Table 5) as well as to toluene. The same concentrations of toluene andbenzene (50 mg/l) resulted in a similar light response. There was noincrease of bioluminescence upon exposure to o-xylene. The lightresponse due to JP-4 was significantly greater than the additiveresponses for JP-4 components (i.e. BTEX compounds) present at theirestimated concentrations (Smith et al., 1981). The increased responsemay be the result of induction due to other components of JP-4 whichwere not tested. A significant light response was observed forethylbenzene after 4 hours. After 2 hours incubation, the cell densitiesfor the ethylbenzene treatments were significantly less than the othersamples, indicating that there may have been a toxicity effect. Otherstudies showed that 50 mg/L ethylbenzene would induce thebioluminescence response without a lag period when cells were previouslygrown on ethylbenzene and then subjected to growing cell assays. TABLE 5EFFECT OF BTEX, PHENOL AND JP-4 CONSTITUENTS ON THE BIOLUMINESCENCERESPONSE OF TVA8 Exposure Time Specific Bioluminescence Treatment^(a)(hours) (namp/OD)^(b) Buffer (Control) 2  0.2 ± 0.1 Toluene 2 291 ± 6 Benzene 2 242 ± 9  Ethylbenzene 2  1.0 ± 0.2 4  47 ± 6^(c) o-xylene 2 0.5 ± 0.1 m-xylene 2 38 ± 3 p-xylene 2 24 ± 2 Phenol 2 70 ± 2 JP-4 2 93± 4^(a)Final concentration for BTEX and phenol treatments was approximately50 mg/L, added as a hydrocarbon-saturated MSM solution. The finalpercentage of water-soluble JP-4 constituents was approximately 2%.^(b)Values are averages ± standard deviation of three replicate samples.Values were normalized to the final cell density (OD₅₄₆).^(c)Value for the 4 hour reading was that measured from a similar butseparate study.

Toluene Growth Comparison of Bioluminescent Reporter with F1

Growth curves for TVA8 and the parent strain, F1 on toluene vapor areshown in FIG. 14. The curves show similar shapes with different lagtimes for TVA8 and F1 which can be attributed to slightly differentinoculum concentrations. Rates were computed from the slopes of thelinear portion of the growth curve for both strains. The average rate ofincrease in optical density for F1 and TVA8, 2.14±0.3 and 2.2±0.3min⁻¹×10⁻³, respectively, were not statistically different (α=0.05).These results demonstrate that the bioluminescence reactions do notappear to affect cell growth.

Bioluminescence of TVA8 was measured during growth on toluene and isshown along with the cell density data in FIG. 15. The bioluminescenceplots show a similar trend to the TVA8 growth curve, although, they areshifted to earlier time points. The graph shows that there is a definitecorrelation between an increase in biomass and an increase in lightproduction. At higher cell densities, cells likely became limited foroxygen resulting in decreased bioluminescence values.

Advantages of the Chromosomally Inserted tod-lux System

The majority of bioluminescent reporters currently being used are theresult of cloning a promoter in front of the promoterless luxCDABE genecassette in pUCD615 and transferring the plasmid construct into thestrain which contained the particular promoter. Plasmid-based systemshave obvious drawbacks such as the need for constant selective pressureto ensure plasmid maintenance as observed by Rice et al. (1995). Anotherimportant consideration is that of plasmid copy number. If the system ispositively regulated, copy number can negatively effect gene expression.Multiple copies of the promoter binding region for the regulatoryprotein on the plasmid compete with the binding site on the chromosomecausing less expression of the operon being studied (Lau et al., 1994).This negative effect is important when using lux fusions for on-linemonitoring of bacterial processes.

Another strategy used in the construction of bioluminescent reporters isthe use of the lux transposon Tn4431 (Shaw et al., 1988). The desiredreporter strain is transposon-mutagenized and constructs are selectedfor bioluminescence upon addition of the specific inducer as in the caseof the nah-lux reporter HK44 (King et al., 1990). However, a problemwith creating a lux fusion by transposon insertion is that the pathwayin which insertion occurs is usually disrupted. For example, in HK44 thelux insertion disrupted nahG (salicylate hydrogenase) and therefore thestrain was no longer able to mediate the complete degradation ofnaphthalene via the nah and sal pathways (Menn et al., 1993). To developa strain for use in monitoring naphthalene degradation, the reporterplasmid had to be conjugated with another strain able to complete themetabolism of naphthalene. Due to these concerns, researchers haveshifted to using transposon delivery systems.

Herrero et al. (1990) constructed a mini-Tn5 delivery system whichconsisted of a mini-Tn5 transposon with unique NotI and SfiI restrictionsites and a pUC derivative containing either of these two restrictionsites flanking the multicloning site. The transposase was provided intrans to provide stability in the final construct. The approach involvedsub-cloning the relevant insert into the particular pUC derivative,cloning it into the mini-Tn5 vector and transposing it into thechromosome of the strain of interest. One drawback to this system isthat it is limited to NotI and SfiI sites and if either of these twoenzymes cut within the insert DNA, alternative strategies have to bepursued. Furthermore, it may be difficult to non-directionally clone alarge DNA fragment such as greater than 12 kbp.

The system described herein is a modification of that describedpreviously. The mini-Tn5 system constructed in this study is based onthe use of five enzymes, AvrII, NheI, SpeI XbaI and NotI, as opposed totwo. Mini-Tn5 KmNX contains unique NotI and XbaI sites which allowdirectional cloning of inserts, negating dephosphorylation of the vectorDNA. The final version of the mini-Tn5 derivative, pUTK211 also containsa strong transcription terminator 5′ to the unique cloning sites toinsure that there is no read-through transcription from a gene in whichthe transposon has been inserted. The cloning vector pLJS used inconjunction with mini-Tn5KmNX allows the utilization of a large regionof the multicloning site flanked by AvrII, NheI, SpeI, XbaI and NotI onone side and AvrII, NheI, SpeI and XbaI on the other. If there is a NotIsite in the fragment to be cloned, the XbaI site can be used fornon-directional cloning. The restriction recognition sequences for theseenzymes are rare (6-base sequences with the exception of NotI whichrecognizes an 8-base sequence). The advantage of the XbaI site is thatit allows the heterologous cloning of AvrII, NheI and SpeI since all ofthese enzymes have the same 5′ overhang, CTAG. This system also allowsthe assembly of larger inserts as seen by the cloning of thetranscription terminator destroying the XbaI site by heterologouscloning using AvrII. The resultant cloning step also regenerated theunique XbaI site. One can use this heterologous cloning strategy ofdestroying and regeneration of the unique XbaI site to assembledifferent DNA fragments for the desired construct. Using this system, P.putida TVA8, a chromosomally-encoded tod-lux bioluminescent reporter wasconstructed.

TVA8 was capable of growing on mineral salts media with toluene orsuccinate demonstrating that the transposon insertion did not disrupt agene necessary for growth. This is a crucial check that must beperformed to ascertain the overall fitness of the strain before furtherevaluation. Furthermore, TVA8 did not show loss of the transposoninsertion or loss of bioluminescence after 100 generations in continuousculture or 5 successive transfers in batch culture. These resultssuggest that selective pressure is not necessary for the integrity ofthe strain. This stability is important since it eliminates the need forantibiotic selection, which if required would exclude the use of thisbioreporter in situ. The strain also was compared to the wild typestrain F1 to ascertain whether or not the bioluminescent reporter was asignificant metabolic drain on the cell, as well as if the site oftransposition was a hindrance to the cell. Growth of TVA8 and F1 ontoluene vapor showed that there was no difference in growth between thetwo strains, suggesting that neither the insertion nor the reporter wasa significant handicap to the cell.

The tod-lux reporter is quite sensitive with a detection limit below 50μg toluene/L. The bioluminescence value at this concentration was80-fold greater than the background bioluminescence level. Thisbioreporter showed a very low background level of bioluminescence (lessthan 1 namp/OD₅₄₆). TVA8 was shown to be useful for quantifying toluenepresent at low concentrations in aqueous solutions. Significant lightlevels were observed for very low optical densities (FIG. 15).

TVA8 may be described as a generalized BTEX bioreporter rather thansimply a toluene bioreporter since it was responsive to benzene,ethylbenzene and m- and p-xylene as well. Since all of these compoundsinduce the bioluminescence response, TVA8 may be used as a bioreporterfor JP-4 jet fuel contamination or presence of any fuel which containsBTEX compounds. The strain may be used for on-line monitoring of TCEcometabolism since the lux and tod operons are under the sameregulation, and the toluene dioxygenase also catabolizes TCE.Bioluminescent reporters may have great potential for field useapplications since they can provide on-line and non-destructive analysesof gene expression as well as detection of chemical contaminants. Thedevelopment of stable transposon insertion of reporter genes intoenvironmental isolates expands the utility of bioreporter strains for insitu sensing of gene expression.

Example 2 Pseudomonas putida B2: A tod-lux Bioluminescent Reporter forToluene and Trichloroethylene Co-Metabolism

The environmental fate and bioremediation potential of trichloroethylene(TCE) have received considerable attention due to its extensiveproduction, use, and occurrence as a groundwater priority pollutant oftoxic and carcinogenic concern. Bacterial metabolism of TCE has beenextensively reviewed (Ensley, 1991). TCE degradation is co-metabolic inthat TCE is not used as a carbon source but is fortuitously degraded.Due to the potential production of carcinogenic vinyl chloride duringanaerobic degradation, much of the recent focus on TCE biodegradationhas been on aerobic, oxygenase-mediated TCE co-metabolism. Substantialinformation has been developed on monooxygenase-mediated co-metabolismof TCE with particular emphasis on the methane monooxygenases and avariety of toluene monooxygenases.

Toluene degradation occurs via catabolic pathways containing bothmonooxygenases and dioxygenases, which have the ability to oxidize TCE.The toluene dioxygenase (todC1-C2BA) contained in Pseudomonas putida F1is also capable of transforming TCE.

Central to the use and further development of aerobic co-metabolic TCEbioremediation is the ability to monitor, control and optimize suchbiodegradative processes. One such strategy has been the development ofbioluminescent lux gene fusions for use in on-line reporter technology(King et al., 1990). The use of lux-reporter systems in the study of theon-line monitoring of naphthalene degradation has been well documented(Heitzer et al., 1995). These reporter systems have also been used toassess the bioavailability of pollutants to catabolic organisms.

This example describes the construction of lux bioreporters formonitoring and optimizing the co-metabolic oxidation of pollutants suchas TCE. For this purpose the tod system contained in P. putida F1 waschosen to develop a tod-lux gene fusion to monitor the expression oftoluene dioxygenase.

Materials and Methods

Strain Construction

The strains and plasmids used in this example are shown in Table 6.Escherichia coli was grown in Luria-Bertani (LB) broth and on LB agarplates at 37° C. Pseudomonas putida F1 was grown on yeastextract-peptone-glucose (YEPG) medium consisting of 0.2 g yeast extract,2.0 g polypeptone, 1.0 g D-glucose and 0.2 g ammonium nitrate (pH 7.0)in 1 L of distilled water at 28° C. TABLE 6 STRAINS AND PLASMIDS StrainPlasmid Relevant Characteristic(s) E. coli JM109 pDTG514 pGem3Z with a2.75-kb EcoR1-SmaI fragment from pDTG350 containing the tod promoter,Amp ® E. coli HB101 pUCD615 Promoterless luxCDABE cassette, mob⁺Amp^(R), Km^(R) P. putida F1 none Contains chromosomally-encoded todoperon for toluene degradation P. putida B2 pUTK30 tod-lux reportercontaining the tod promoter fragment inserted upstream of thepromoterless luxCDABE cassette, Amp^(R), Km^(R) E. coli DF1020 pRK2013Helper plasmid, Amp^(R), Km^(R), Tra⁺

One-liter cultures of E. coli JM109 and HB101 harboring the appropriateplasmids were harvested and plasmid DNA was isolated using a modifiedalkaline lysis procedure (Promega, 1992). The plasmid DNA was subjectedto CsCl density gradient purification, followed by butanol extractionand ethanol precipitation (Sambrook et al., 1989). Plasmid DNA wasresuspended in TE buffer (10 mM Tris-base, 1 mM EDTA, pH 8.0) and storedat 4° C. until used. Restriction endonucleases and T4 DNA ligase wereobtained from Gibco BRL (Gaithersburg, Md.) and used according tomanufacturers' protocols. Cloning techniques were performed as outlinedin Sambrook et al. (1989). The reporter plasmid pUTK30 was generated bycloning the tod promoter (Lau et al., 1994; Wang et al., 1995) frompDTG514 (Menn et al., 1991) in front of the lux gene cassette of pUCD615(Rogowsky et al., 1987). This was accomplished by directionally cloninga 2.75-kb EcoR1-XbaI fragment from pDTG514 into an EcoR1-XbaI digest ofpUCD615 (FIG. 16). Transformations were performed using subcloningefficiency competent cells (Gibco BRL, Gaithersburg, Md.) according tothe manufacturer's protocol. Transformants were selected on LB platescontaining 50 μg ml⁻¹ kanamycin. Plasmid minipreps of transformants wereperformed as described by Holmes and Quigley (1981) and cleaved withBamHI to confirm insertion of the tod fragment. The resultant E. colistrain, JBF-7 harbored the reporter plasmid pUTK30.

Triparental matings were carried out using a modified version of thefilter technique. Pure cultures of donor (JBF-7, pUTK30), helper(DF1020, pRK2013; Figurski and Helsinki, 1979), and recipient (F1) weregrown to an optical density at 546 nm (OD₅₄₆) of approximately 1.0 in LBbroth with appropriate antibiotics. Cells were harvested bycentrifugation at 9800×g for 10 min. The pellets were suspended andwashed three times in 100 ml 50 mM KH₂PO₄ (pH 7.0), and suspended in 50ml 50 mM KH₂PO₄.

The three strains were mixed using a ratio of 2:1:1(donor/helper/recipient). The cell suspension as filtered through aTeflon membrane (47 mM, 022 μm pore size) and the filter was placed on aLB plate. After overnight incubation at 28° C., the filter was removedand washed in 1.5 ml 50 mM KH₂PO₄. Serial dilutions were performed anddilutions were plated onto Pseudomonas Isolation Agar plates (Difco,Detroit, Mich.). After a 48-hour incubation, toluene vapor wasintroduced and colonies which produced light were selected for furthercharacterization. One of five kanamycin-resistant strains which emittedlight in response to toluene vapor, P. putida B2, was chosen for use inthe remaining studies.

Bioluminescence Analysis

In the batch and reactor studies, bioluminescence was measured using aphotomultiplier, which converts the light to an electric current. Thephotomultiplier in the resting cell assays and the reactor system wasconnected to a computer and bioluminescence as namps current wasrecorded.

Batch Studies

Assays of growing cells were conducted as described by Heitzer et al.(1992). An overnight culture from a frozen stock of P. putida B2 wasprepared in a 250-ml Erlenmeyer flask containing 100 ml LB and 50 μgml⁻¹ kanamycin. A subculture was prepared and cells were used in mid-logphase (OD₅₄₆ of 0.45-0.48). A 2.5-ml aliquot was added to 2.5 ml mineralsalts medium (MSM) containing 0-50 mg L⁻¹ toluene or 10-100 μl of JP4jet fuel-saturated MSM. The concentration of toluene in water saturatedwith JP4 jet fuel is approximately 8 mg L⁻¹ (Smith et al., 1981).Bioluminescence was measured every 30 min.

Cells for resting cell assays were grown on MSM supplemented with 2.7 gL⁻¹ succinate. A culture of P. putida B2 was harvested at on OD₅₄₆ ofapproximately 0.8. The cells were centrifuged at 15000×g for 10 min, andresuspended in MSM to OD₅₄₆ of 0.6 four milliliters of culture wereadded to each of six 26-ml vials with Mininert valves (Dynatech,Chantilly, Va.) with stir bars. One vial was used for multiple tolueneexposures, while the remainder were used for single exposures. The vialswere magnetically stirred in a light-tight sampling cell.Toluene-saturated MSM and MSM alone were added to yield an OD₅₄₆ of0.47, and 10 mg L⁻¹ toluene was injected six times over a 130-h periodof the multiple-exposure vial. At the same time points, asingle-exposure vial was injected with 10 mg L⁻¹ toluene. The lightresponse was recorded every 3 min with a photomultiplier connected to adata acquisition computer.

Immobilized Cell Reactor System

P. putida B2 was encapsulated in alginate beads for the immobilized cellreactor system. Cells were grown in 1 L LB to an OD₅₄₆ of 1.2 and werecentrifuged at 5500×g for 10 min, washed three times in 0.9% NaCl andsuspended in 40 ml 0.9% NaCl. The cell suspension was added to 80 ml ofan alginic acid solution (28 g L⁻¹ low viscosity alginate, 0.9% NaCl)(Webb, 1992). The cell-alginate suspension was placed in a 60-mlsyringe, forced through a 25-gauge needle, and allowed to drop into a0.5 M CaCl₂ solution. The alginate was cross-linked by the Ca²⁺ ions,thus encapsulating the cells. The cells were subsequently placed in afresh solution of 0.1 M CaCl₂ and allowed to sit for 30 min prior touse.

A differential volume reactor (DVR) was used to simulate a section of anideal plug flow reactor. Influent to the reactor was dispersed through aporous metal frit to provide a flat velocity profile to the bed. Thereactor measured 5.0 cm i.d.×5.0 cm long. A complete description of thisreactor can be found in Webb et al. (1991). In this investigation, asystem was designed incorporating the DVR as illustrated in FIG. 17. Thesystem was equipped with three Millipore (Bedford, Mass.) stainlesssteel substrate containers rated to 690 kPa. The feed from the substratevessels to the reactor inlet was controlled by two Filson (Middleton,Wis.) 301 HPLC pumps. A flow rate of 0.4 ml min⁻¹ was maintained. Thesubstrate vessels were pressurized with oxygen to provide the systemwith an electron acceptor. All medium vessels contained trace mineralmedium (Lackey et al., 1993) with the addition of 3 mg L⁻¹ pyruvic acidand a 0.1 M solution of Tris-base (pH 7.0). phosphate buffers were notused since phosphate ions complex with Ca²⁺ ions and disrupt withalginate crosslinking. In addition to this medium, two of the vesselscontained either TCE or toluene. The inlet concentration of toluene wasaltered by using square-wave perturbations with 20-h cycles (10 h withtoluene, 10 h without toluene) using an HPLC pump controlled by a timer.During feed portions of the cycle, 10 mg L⁻¹ toluene was introduced intothe inlet of the reactor. The inlet TCE concentration was constant at 20mg L⁻¹.

TCE and Toluene Analysis

Analysis of TCE in the reactor effluent was performed online using astripping column (12.5 cm length and 0.4 cm inner diameter) packed with3-mm glass beads to provide adequate surface area for TCE separation.TCE was stripped with helium, the GC carrier gas. The stripping columnoutlet was attached to a gas chromatograph (GC, Hewlett Packard(Wilmington, Del.) (HP) 5890 Series II) with an electron capturedetector by a heated sample line maintained at 75° C. Automaticinjections (25 μl) were made by a computerized control process (HP ChemStation software). The GC was equipped with a cross-linked methylsilicone capillary column (length 30 m, i.d. 0.2 mm, 0.33-μm filmthickness) while the oven was operated isothermally at 60° C. Otheroperating parameters included an injection temperature of 150° C.,detector temperature of 200° C. and a split ratio of 10:1. This systemwas equipped with a bypass line around the reactor in order to calibratethe stripping column.

Toluene samples were removed at 0.5-ml aliquots from the effluentsampling port (FIG. 17) and injected into 1.5 ml sample vials. Headspaceanalysis was performed using a Shimadzu (Columbia, Md.) GC-9A gaschromatograph equipped with a 2.44-m, 3.2-mm diameter Poropak N packedcolumn and a flame ionization detector. The isothermal temperature ofthe oven was 210° C., and both the detector and injector temperatureswere 220° C.

Results

Batch Studies

Assays of growing cells showed an increasing bioluminescent responsewith increasing concentrations of toluene, up to 10 mg L⁻¹ toluene,after 90 minutes exposure (FIG. 18). The relationship was linear overthis range. The bioluminescent response varied from 2.4 namp at 0.1 mg/ltoluene to approximately 90 namp for 10, 20 and 50 mg/l toluene. Therewas not a significant bioluminescent response for 0 and 0.01 mg/ltoluene. Similarly, the light response increased with increasingconcentration of water-soluble jet fuel components (FIG. 18). At 10 μljet fuel-saturated MSM added (approximately 0.02 mg/l toluene), thelight response was 16 namp, while at 100 μl added (approximately 0.2mg/l toluene). The response increased to 31 namp. The bioluminescenceresponse for the 0.1 mg/l toluene equivalent of jet fuel was about 10times that for 0.1 mg/l toluene, so other components beside tolueneappear to have affected bioluminescence.

In resting cell assays, the bioluminescent response to single exposuresof toluene was rapid and reproducible (FIG. 19). The initial injectionto the multiple exposure vial showed the same characteristic lightresponse as each single exposure vial. However, there was a slowerresponse (the rate of increase in bioluminescence, H⁻¹) upon initialexposure to toluene compared with the response of cells previouslyexposed to toluene. In addition, the response rate increased with eachexposure to toluene (Table 7). However, the maximum bioluminescentresponse for both the single and multiple exposures was the same at573±127 namp. TABLE 7 BIOLUMINESCENT RESPONSE RATE (NAMP/HR) FORMULTIPLE AND SINGLE EXPOSURES OF 10 MG/L TOLUENE^(A) Time Point MultipleExposure Vial^(b) Single Exposure Vials^(c) 1 95 ND 2 321 137 3 642 67 4768 60 5 737 60 6 973 49^(a)Response rate is defined as rate of bioluminescence increase withtime.^(b)A single vial, with multiple additions of toluene.^(c)A new vial, previously unexposed to toluene, injected with tolueneat each time point.ND, not done.

Immobilized Cell Reactor System

The DVR system loaded with alginate-encapsulated P. putida B2 was usedto determine the light response and TCE co-metabolism of P. putida B2when exposed to toluene in immobilized systems. Experimental resultsshowed a rapid bioluminescent response under the introduction oftoluene. FIG. 20 shows light response of the reporter strain in thereactor to the change in inlet toluene concentration and removal of TCE.The data show a direct response of bioluminescence with respect totoluene concentration. During the cycle, light emission increased by16.3±1.2 namp/hr. The toluene effluent concentration approached zeroafter the toluene feed was stopped, and the light response in thereactor decreased at a rate of 3.4±0.8 namp/hr. A direct correlationbetween bioluminescence and TCE degradation was observed. The maximumlight response was 43.4±6.8 namp. The steady-state TCE effluentconcentration when toluene was being introduced into the system was16.5±0.2 mg/l (20% removal), while the effluent toluene concentrationwas 5.8±0.1 mg/l (50% removal). This represents a ratio of 1.7 μmoltoluene degraded/μmol TCE degraded. While results from the differentassay types showed similar response to toluene, the magnitude ofbioluminescence cannot be compared due to several differences betweenexperimental conditions (i.e., sample agitation, cell physiology, lightmonitoring).

Discussion

Assays of growing cells demonstrated not only a qualitativebioluminescent response to toluene, but a quantitative response as well.There was a linear relationship between bioluminescence and tolueneconcentration between 0 and 10 mg/l in assays of growing cells. Inaddition, the bioluminescent response was proportional to dilutions of acomplex environmentally relevant contaminant, jet fuel. However, themagnitude of the bioluminescent response to jet fuel was higher thanwould be expected if the response was due solely to the toluene in thejet fuel. Work with another bioluminescent strain has recently shownthere is a significant bioluminescent response to solvents (Heitzer etal., 1996). It was demonstrated that cells were limited for the aldehydesubstrate of the luciferase reaction. It was hypothesized that solventsperturb the cellular membrane, causing intracellular concentrations offatty acids to increase. Since fatty acids are reduced to thecorresponding aldehydes by the lux enzymes, increased amounts wouldnegate the aldehyde limitation, causing higher bioluminescence. Thissolvent effect might explain the observed difference in magnitude ofbioluminescence between pure toluene and toluene in a solvent matrix inassays of growing cells.

Typically, in the environment, cells would not be in midlog phase ofgrowth. Therefore, the inventors examined the bioluminescent propertiesunder resting cell conditions as well. Even in cells with toluene as anintermittent sole carbon source, the bioluminescent response wasreproducible for at least 5 days. A more rapid bioluminescent responsewas observed for cells previously exposed to toluene, but the maximumbioluminescence remained constant.

Immobilized P. putida B2 allowed on-line monitoring of degradativeactivity towards toluene and TCE in a DVR. The system showed a directcorrelation between toluene degradation and bioluminescence. Because thelux and tod operons are under the same promoter control, bioluminescenceindicated that the tod operon was expressed, and TCE was co-metabolized.Therefore, there was a direct mechanistic correlation betweenbioluminescence and TCE co-metabolism. In this study, TCE did not appearto induce the tod operon in P. putida B2 as was reported for another P.putida strain (Heald and Jenkins, 1994). FIG. 20 shows that in theabsence of toluene, TCE influent and effluent concentrations wereequivalent and there was no bioluminescence increase.

Exposure to TCE and/or its metabolites may be toxic and may affectdegradative enzyme activity. However, the intensity of bioluminescencewas reproducible in successive perturbations of toluene even in thepresence of TCE (FIG. 20). These data showed the tod-lux reporterprovided an on-line measurement of tod gene expression, and alsoprovided an indication of potential toxic effects due to continuous TCEexposure. At 20 mg/l TCE, there did not appear to be any toxic effects.This example demonstrated that there is a distinct and reproducibleresponse to toluene under a variety of physiological conditions (growingand resting free cells and immobilized cells).

Example 3 Kinetics and Response of a P. fluorescens Biosensor

Polycyclic aromatic hydrocarbons (PAH) are persistent environmentalcontaminants that are toxic and carcinogenic. Hundreds of sites existnationwide that are highly contaminated at concentrations greater thangrams PAH per kilogram of soil. These sites range from 1 to over 100acres. Indigenous soil organisms have demonstrated their ability todegrade these compounds.

King et al. (1990) reported the construction of pUTK21 by thetranscriptional fusion of the luxCDAB cassette and the nahG gene withinthe archetypal NAH plasmid pKA1. The catabolic plasmid pKA1 from whichpUTK21 was engineered is organized in two operons, the naphthalene andsalicylate operons, and mediates the degradation of naphthalene,salicylate, and many other pollutants. The pUTK21 contains two pathways,an upper pathway, which codes for the degradation of naphthalene tosalicylate (the naphthalene operon), and a lower engineered pathway,which codes for the lux pathway. The lower pathway no longer codes forsalicylate degradation as the nahG gene was disrupted by insertion ofthe luxCDABE cassette. Both pathways of pUTK21 are controlled bypromoters induced by salicylate. The reporter bacterium, Pseudomonasfluorescens 14K44 (HK44), harbors the pUTK21. The HK44 supplements thedisabled salicylate operon by naturally degrading salicylate by apathway independent of nah. A positive-quantitative relationship betweenbioluminescence and inducer concentration (naphthalene and salicylate)as well as degradation of these compounds was demonstrated (DiGrazia,1991). Bioluminescence activity requires oxygen, NADPH, ATP, FMNH₂, andaldehyde substrate. The luxCDABE genes code heterodimeric luciferase,reductase, transferase, and synthetase. The light reaction requires along-chain aldehyde substrate, which is converted to a fatty acid duringthe light reaction. The fatty acid reductase complex (reductase,transferase, and synthetase) is essential as it regenerates thelong-chain aldehyde substrate from the fatty-acid product.

Previous studies employing free HK44 indicate a linear light responsewith salicylate and naphthalene concentration (Heitzer et al., 1994).This example describes the form and parameters of salicylate byimmobilized HK44. Potential differences exist in bacterial physiologybetween free and immobilized states. Because they are “noninvasive,nondestructive, rapid, and population specific”, bioluminescent reporterstrains have the potential to rapidly indicate bioavailability,degradative activity, and optimal degradation conditions in situ. TheHK44 biosensor described herein may be produced by immobilizing HK44 ona light-culminating device (e.g., fiber optic). The HK44 sensor couldthen be employed to continuously monitor conditions and degradation insoils. Uses of such a sensor could include (1) detection of plumes(e.g., salicylate is a mobile daughter compound produced by thebiological degradation of naphthalene and several other PAH) or (2)monitoring remediation during later stages of remediation as PAHconcentrations are reduced. Provided are mathematical descriptions ofsalicylate degradation by immobilized HK44. An exemplary system is shownwhich has a packed-bed reactor (PBR) with alginate-immobilized HK44.

Mathematical Models

This example describes a plug-flow reactor with a bed of immobilizedHK44. Assuming that significant flow occurs only in the axial direction,an unsteady-state shell mass balance on the bulk liquid phase from timet to t+Δt and from position z to z+Δz results in: $\begin{matrix}{{{\int_{t}^{t + {\Delta\quad t}}{\left\lbrack {\in {{S\left\lbrack {C_{i}\overset{\_}{V}} \right\rbrack}_{z,t} -} \in \quad{{S\left\lbrack {C_{i}\overset{\_}{V}} \right\rbrack}_{{z + {\Delta\quad z}},t} +} \in {{S\left\lbrack {{- D_{i}}\frac{\partial C_{i}}{\partial z}} \right\rbrack}_{z,t} -} \in {S\left\lbrack {{- D_{i}}\frac{\partial C_{i}}{\partial z}} \right\rbrack}_{{z + {\Delta\quad z}},t}} \right\rbrack{\mathbb{d}t}}} - {\int_{z}^{z + {\Delta\quad z}}{{N_{P_{i}}\left( {{1 -} \in} \right)}S\quad{\mathbb{d}z}}}} = {\int_{z}^{z + {\Delta\quad z}}{\left\lbrack {S \in {C_{i}{_{{t + {\Delta\quad t}},z}{{- S} \in C_{i}}}_{t,z}}} \right\rbrack\quad{\mathbb{d}z}}}} & (24)\end{matrix}$

Equation (24) reduces to Equation (25) by applying the mean valuetheorem of integral calculus, dividing through by Δz and Δt, takinglimits as Δt and Δz go to 0, and substituting for the rate of masstransfer, the surface area of a spherical bead, and the superficialvelocity: $\begin{matrix}{{{{- \overset{\_}{V}}\frac{\partial C_{i}}{\partial z}} + {D_{i}\frac{\partial^{2}C_{i}}{\partial z^{2}}}} = {\frac{\partial C_{i}}{\partial t} + {\frac{\left( {{1 -} \in} \right)}{\in}K_{i}\frac{3}{r_{0}}\left( {{C_{i} - C_{p_{i}}}❘_{r = r_{0}}} \right)}}} & (25)\end{matrix}$

-   -   Equation (18) can be made dimensionless by the following        substitutions:        C_(i) _(o) CDPi=C_(pi),C_(i) _(o) C_(D) _(i) =C_(i), C_(i) _(o)        θ_(si)C_(Dsi)=C_(S) _(i) LΦ=z, t _(r) τ=t, and r ₀ φ=r  (26)    -   Equation (20) results upon substitution: $\begin{matrix}        {{{\frac{t_{r}{D_{P}}_{i}}{L^{2}}\frac{\partial^{2}C_{D_{i}}}{\partial\Phi^{2}}} - \frac{\partial C_{D_{i}}}{\partial\Phi} - \frac{\partial C_{D_{i}}}{\partial\tau} + {\frac{\left( {{1 -} \in} \right)}{\in}\frac{3t_{r}}{r_{0}}{K_{i}\left( {{C_{D_{i}} - C_{P_{i}}}❘_{r = r_{0}}} \right)}}} = 0} & (27)        \end{matrix}$

The initial condition assumes a clean bed. The boundary conditions indimensionless form are $\begin{matrix}{{\frac{\partial C_{D_{i}}}{\partial\Phi}❘_{\Phi = 1.0}} = {{{0\quad{and}{\quad\quad}C_{D_{i}}}❘_{\Phi = 0}{{{- C_{D_{i}}} + {\frac{D_{i}}{\overset{\_}{V}L}\frac{\partial C_{D_{i}}}{\partial\Phi}}}❘_{\Phi = 0}}} = 0}} & (28)\end{matrix}$

-   -   An unsteady-state mass balance on the solid phase yields:        $\begin{matrix}        {{\int_{t}^{t + {\Delta\quad t}}{\left\lbrack {{4\pi\quad{r^{2}\left( {\in_{p}N_{p_{i}}} \right)}}❘_{r,t}{{{- 4}{\pi\left( {r^{2} + {\Delta\quad r}} \right)}\left( {\in_{p}N_{p_{i}}} \right)}❘_{{r + {\Delta\quad r}},t}{{+ 4}\pi\quad r^{2}\Delta\quad{rR}_{i}}}} \right\rbrack\quad{\mathbb{d}t}}} = {\int_{r}^{r + {\Delta\quad r}}{\left\lbrack {\left( {\in_{p}{C_{p_{i}} + C_{s_{i}}}} \right)❘_{r,t}{{- \left( {\in_{p}{C_{p_{i}} + C_{s_{i}}}} \right)}❘_{r,{t + {\Delta\quad r}}}}} \right\rbrack\quad{\mathbb{d}r}}}} & (29)        \end{matrix}$        where liquid diffusion rate is assumed to be dominant and        equilibrium is assumed in the pores. A term can be developed for        the absorbed solid-phase flux by adding terms to the above        development. Equation (30) results after applying the mean value        theorem of integral calculus, dividing by Δz and Δt, taking        limits as Δt and Δz go to zero, making the substitutions in        Equation (26), and assuming linear adsorption: $\begin{matrix}        {{{\frac{\in_{p}{D_{p_{i}}t_{r}}}{r_{0}^{2}}\frac{\partial^{2}C_{D_{p_{i}}}}{\partial\varphi^{2}}} + {\frac{2 \in_{p}{D_{p_{i}}t_{r}}}{r_{0}^{2}\varphi}\frac{\partial C_{D_{p_{i}}}}{\partial\varphi}} + {\frac{t_{r}}{C_{i_{0}}}R_{i}}} = {\frac{\partial D_{D_{p_{i}}}}{\partial\tau}\left( {\in_{p}{{+ \theta_{s_{i}}}\frac{\mathbb{d}C_{D_{s_{i}}}}{\mathbb{d}C_{D_{p_{i}}}}}} \right)}} & (30)        \end{matrix}$

The exact form of Ri is unknown for this system. The Michaelis-Mentenreaction model (MMRM) is general and reflects a nonlinear relationshipbetween degradation rate and substrate concentration rate and substrateconcentration. This nonlinear relationship arises from the finitedegradative capacity of biological systems. At low concentrations, theMMRM approaches a reaction rate which is first order in substrateconcentration. As the degradative capacity of the system is approachedor exceeded, the MMRM becomes zero order in concentration. Thus a largenumber of conditions distributed over the reaction regime are requiredto properly measure the two MMRM rate constants. The reaction rate formand constants were elucidated by first comparing the steady-statebehavior of the HK44 to the limiting cases of the MMRM. These limitingcases were represented mathematically as first order in salicylate andfirst order in biomass as:−R _(salicylate) =K ₂ C _(Biomass) C _(Salicylate)  (31)

-   -   and as zero order in concentration and first order in biomass        as:        −R _(salicylate) =K ₁ C _(Biomass)  (32)

Equations (31) and (32) each require a single rate constant instead oftwo as required by the Michaelis-Menten model. Practically, the linearrate models, Equations (31) and (32), provide a more stringentdescription of behavior than the two-parameter, nonlinear MMRM.

Initial and boundary conditions assume a clean bed, symmetry within thebead, and no accumulation at the solid interface: $\begin{matrix}{{{C_{D_{p_{i}}}\left( {0,\varphi} \right)} = 0},{\frac{\partial C_{D_{p_{i}}}}{\partial\varphi} = 0},{{{{and}\frac{\in_{p}D_{p_{i}}}{r_{0}K_{i}}\frac{\partial C_{D_{p_{i}}}}{\partial\varphi}}❘_{\supset {= 1}}} = \left( {{C_{D_{i}} - C_{D_{p_{i}}}}❘_{\varphi = 1}} \right)}} & (33)\end{matrix}$

Equations (27) and (30) indicate that the processes which affect thedistribution and conversion of the substrate include: (1) dispersion andconvective transport in the bulk phase; (2) bulk and internalsolid-phase mass transfer resistance; (3) adsorption onto the alginateinside the bead pores; and (4) chemical conversion by bacteria onlywithin the bead. A constant distribution of biomass is assumed with nogrowth. If growth occurs, then biomass distribution may become afunction of bead radius (Kuhn et al., 1991). The model was simplifiedfor analyzing bed steady-state behavior by assuming negligible masstransfer resistance. This simplified model, when combined with thereaction rate Equation (31), has the solution (Danckwerts, 1953):$\begin{matrix}{{\frac{C_{Salicylate}}{C_{{{Salicylate}❘\Phi} = 0}} = \frac{4\eta\quad{\exp\left( {{Pe}/2} \right)}}{{\left( {1 + \eta} \right)^{2}{\exp\left( {{Pe}\quad{\eta/2}} \right)}} - {\left( {1 - \eta} \right)^{2}{\exp\left( {{- {Pe}}\quad{\eta/2}} \right)}}}}{{where}\text{:}}} & \left( {34a} \right) \\{\eta = \left( {1 + \frac{4K_{x}C_{Biomass}}{\overset{\_}{V}{Pe}}} \right)^{1/2}} & \left( {34b} \right)\end{matrix}$

This simplified model, when combined with the reaction rate Equation(33) has the solution: $\begin{matrix}{{C_{Salicylate} = {\frac{{\vartheta exp}\left\lbrack {{Pe}\left( {\Phi - 1} \right)} \right\rbrack}{Pe} - {\vartheta\Phi} - \frac{\vartheta}{Pe} + 1}}{{where}\text{:}}} & \left( {35a} \right) \\{\vartheta = {\frac{{Lk}_{1}}{\overset{\_}{V}}\frac{C_{Biomass}}{C_{Salicylate}❘_{\Phi = 0}}}} & \left( {35b} \right)\end{matrix}$

The unsteady-state behavior and the full model predictions could then becompared for evaluation of critical assumptions. Mathematical problemsfor which analytical solutions were unavailable were solved using thePDECOL software package (Madsen and Sincovec, 1979). Availableanalytical solutions and numerical results were compared for diffusion,transport, and reaction processes (Crank, 1975). Other investigatorshave also used PDECOL for simulation of PBR processes (Costa andRodrigues, 1985).

Materials and Methods

A previously developed PBR system with on-line instrumentation (Webb,1992; Webb et al., 1991) was used to measure bacterial degradative andbioluminescent activity under conditions mimicking those of thesubsurface. The reactor design is detailed in FIG. 21. The PBR wastemperature controlled and fitted with metal frits welded to the inletand outlet to retain and distribute feed to immobilized cultures. Anadditional inter-cavity insert allowed the installation of filters(e.g., 0.2-mm inorganic and polymeric) between the packed bed and outletfrit that filtered the effluent for automatic injection into the on-linehigh-performance liquid chromatography (HPLC) and that produced auniform resistance to flow that improved the overall distribution to thereactor (Webb, 1992). The reactor had an internal diameter of 1.34 cm,whereas the bed length could be varied from 0.8 to 11.0 cm. HPLC pumpssupplied media to each reactor from pressurized feed reservoirs. Allnutrients, including oxygen, entered the reactor dissolved in the liquidphase. Naphthalene and salicylate were detected with an on-line HPLCusing a Vydac TP20154 column (Sep/A/Ra/Tions Group, Hesperia, Calif.)with a FL-4 dual-monochromator fluorescent detector (Perkin-Elmer,Norwalk, Conn.). Respective excitation and emission wavelengths forsalicylate detection were 290 and 360 nm. The reactor was fitted with anon-line light detector for monitoring bioluminescent activity (Dunbar,1992) consisting of a glued fiber bundle (Ensign Bickford Optics,Simsbury, Conn.) placed approximately 2.5 cm from the entrance of thereactor. An Oriel Inc. (Stratford, Conn.) 7070 photomultiplier detectionsystem using a Model 77348 photomultiplier tube (radiant sensitivity of80 MA/W near 500 nm) were employed.

A second apparatus simulated flow passed through an alginate biosensor.The apparatus was composed of a flow cell and a Hamamatsu photodiode(Bridgewater, N.J.) with an attached layer of HK44 immobilized inalginate. The flow cell volume was 5 ml, whereas the flow rate wasmaintained at 1.5 ml/min. The concentration of salicylate was variedwhile the light was monitored with the photodiode. Mineral salts mediawas used for these experiments with varying concentration of inducer.

Mineral salts media (pH 7.2) consisting of MgSO₄ 7H₂O (0.1 g/l), NH₄NO₃(0.2 g/l), trizma™ base (3.03 g/l), MgO (1.0×10⁻³ g/l), CaCl₂ (2.9×10⁻⁴g/l), FeCl₃.6H₂O (5.4×10⁻⁴ g/l), ZnSO₄.7H₂O (1.4×10⁻⁴ g/l), CuSO₄(2.5×10⁻⁵ g/l), H₃BO₄ (6.2×10⁻⁶ g/l), and Na₂MoO₄.H₂O (4.9×10⁻⁵ g/l)were supplied to the reactor with an appropriate carbon source fordegradation and bioluminescence studies. Kinetic bacterial studies werephosphate limited and maintained aerobic by con-trolled pressurizationof feeds and the reactor.

The HK44 was prepared for immobilization by adding freshly thawedinoculum (frozen at −70° C. until use) to 100 mL of YEPG media whichcontained glucose (1.0 g/l), polypeptone (2.0 g/l), yeast extract (0.2g/l), NH₄NO₃ (0.2 g/l), and tetracycline (14 mg/l). The culture wasshaken for 15 hours at 25° C. and then centrifuged at 8000 rpm for 10minutes. The pellet was then washed with 0.9% NaCl solution three timesbefore immobilization. The concentration of HK44 was enumerated bymeasuring optical density at 546 nm. The HK44 was immobilized bysuspending bacteria in 0.9% NaCl solution and mixing with alow-viscosity alginate (28 g/L) and NaCl (9 g/L) solution in a ratio of1:2 by volume. Beads were formed by controlled dropwise addition to 0.1M CaCl₂ solution by a syringe needle installed in an air jet controlledby a precision regulator (Porter Instrument Co., Hatfield, Pa.) and apiece of 0.003-in. (i.d.) tubing. Beads diameters were measured usinggentle wet sieving. Alginate beads were dissolved in 50 mM sodiumhexametaphosphate.

Naphthalene and sodium salicylate adsorption isotherms on calciumalginate were measured using batch equilibrium and breakthrough curvemethods (Ruthyen, 1984). Residence time distributions for dispersion andliquid-phase mass transfer measurements were evaluated using salicylate(0.1 M), potassium, and/or bromide (1.0 M) introduced through a six-portHPLC valve up-stream of the reactor. Tracer concentrations were measuredusing a Waters ion-chromatography system with a series 510 HPLC pump,IC-PAK anion exchange column, and 431 conductance detector (Waters,Cam-bridge, MA) or monitored continuously using fluorescence.

Abiotic Properties

Typically, almost all of the bead diameters ranged between 3.9×10⁻² and7.5×10⁻² cm. For example, 4, 65, and 31 wt % were retained on7.5×10^(−2, 4.5×10) ⁻², and 3.9×10⁻² cm screens, respectively.Salicylate did not measurably absorb onto the alginate, whereas thenaphthalene isotherm was linear with a dimensionless ratio of 8.4 (FIG.22). Final equilibrium liquid-phase concentration ranged from 0.0 to1.5×10⁻⁴ M, whereas solid concentrations ranged to 1.1×10⁻³ moles ofnaphthalene per liter of alginate. Dispersion was measured bylinearizing the analytical relationships derived by Haynes and Sarma(1973). Slopes ranged between 1×10⁻² and 2×10⁻² m²/min, indicating thatdispersion was on the order of 10⁻² cm²/min. Comparison of reactorvolumes and average residence times demonstrated that channeling was notsignificant. Hydrophobic filters effected a uniform resistance to flowover the reactor outlet and greatly improved flow characteristics. Blotnumbers ranged around 100 and indicate that mass transfer was notsignificant.

Alginate appears to be a good immobilization media for naphthalene andsalicylate reporter bacteria due to its favorable transport andadsorption properties. Also, alginate may be formed into shapes usefulfor sensor applications (e.g., as a thin sheet attached directly to alight probe). Salicylate and naphthalene are good test compounds becausenaphthalene is an abundant environmental pollutant and generally foundwith other PAH contaminants, whereas salicylate is a metabolite ofnaphthalene and several other PAH. Care should be taken in choosing animmobilization matrix for detecting other PAH, because larger PAH maysignificantly deviate from these model compounds in their solubility andabsorption characteristics.

Kinetic Evaluation of P. fluorescens HK44

Different combinations of feed concentrations, biomass, and flow rates,listed in columns 2 to 4 of Table 8, were varied in 18 studies todetermine the form of the degradation rate equation and associatedconstants. Five charges of immobilized cells used for these studies areindicated by a number designation in column 1. Liquid-phase masstransfer was estimated using the correlation of Kataoka et al. (1973).Steady-state behavior was achieved within several bed volumes after aperturbation, consistent with predictions by Equations (20) and (23)using measured parameters. Bacterial degradative activity was thenconstant, although a small amount of drift was noticed in some studies(indicated by higher standard deviations). Ranges for biomass,salicylate concentration, and residence time were 1.9×10⁹ to 3.5×10⁹cells/ml, 2.25 to 4.5 mg/l, and 14 to 150 min, respectively. Salicylatewas supplied to the reactor at or below 4.5 mg/l. Stoichiometric levelsof dissolved oxygen might prove toxic to HK44 at high salicylateconcentration. Concentrations above this range would probably not berealistic as very few PAH are soluble at greater than 1-mg/lconcentrations (Lee et al., 1979). Salicylate conversion, listed incolumn 5, ranged from 9% to 92%, with standard deviations ranging from1.08% to 2.96% normalized to the effluent concentrations. Standarddeviations and average conversions in column 5 were calculated using anaverage of 100 data points for each case.

Five independent studies were repeated and demonstrated reproduciblerelationships between substrate conversion, biomass, feed concentration,and re-actor residence time: (1) studies 4 b and 3 d had conversions of0.62; (2) studies 4 f and 4 c had con-versions of 0.50; (3) studies 3 eand 4 d had respective conversions of 0.41 and 0.39; (4) studies 1 a and1 c had respective conversions of 0.64 and 0.73; and (5) studies 3 c and3 f had respective conversions of 0.77 and 0.70.

Equation (27), using the degradative rate constant as the soleadjustable parameter, provided a good description of the experimentaldata. As depicted in FIG. 23 and FIG. 24, the reaction rate decreasedwith decreasing substrate concentration. The regressed degradation rateconstants from experimental sets 1 a-c, 3 a-f, and 4 a-f were tightlygrouped. The rate constants obtained using the full data set and subsetswere: (1) 2.23×10⁻² dm³ g⁻¹ min⁻¹ fitted to the complete set; (2)1.88×10⁻² fitted to subset 1 a-c; (3) 2.85×10⁻² fitted to subset 2 a-c;(4) 2.06×10⁻² fitted to subset 3 a-f; and (5) 2.28×10⁻² fitted to subset4 a-f. FIG. 23 and FIG. 24 demonstrate good agreement between the fulldata set and Equation (27) using 2.23×10⁻². Study 2 a-c contributed themost to the total residual for all 18 studies. DiGrazia (1991) foundthat naphthalene degradation by HK44 could be described by a rate termwhich was first order in naphthalene and first order in biomass.

Equation (28) was less appropriate for describing the kinetics of HK44than Eq. (X). Residuals were generally an order of magnitude larger thanthose using Equation (27). Also, the fundamental relationships suggestedby Eq. (X) were not born out by the data. The rate constants obtainedusing Eq. (28) ranged over almost half an order of magnitude, 4.33×10⁻⁵to 1.35×10⁻⁴ min⁻¹. TABLE 8 STEADY-STATE DEGRADATION AND LIGHTPRODUCTION BY P. FLUORESCENS HK44 AS A FUNCTION OF BIOMASS, FEEDCONCENTRATION, AND RESIDENCE TIME Sodium Biomass salicylate Salicylateeffluent ± σ Experiment (cells/mL) (mg/L) τ (min) (mg/L) Bioluminescence± σ-(nA) 1a 3.3 × 10⁹ 2.25 14 1.43 ± 2.85 × 10⁻² Unavailable 1b 3.3 ×10⁹ 2.25 58 0.46 ± 0.84 × 10−² Unavailable 1c 3.3 × 10⁹ 2.25 14 1.65 ±4.77 × 10⁻² Unavailable 2a 3.5 × 10⁹ 2.47 24 1.03 ± 2.83 × 10⁻² 2.71 ±4.0 × 10⁻² 2b 3.5 × 10⁹ 2.47 26 0.86 ± 0.93 × 10⁻² 1.81 ± 2.0 × 10⁻² 2c3.5 × 10⁹ 2.47 94 0.20 + 0.28 × 10⁻² 0.16 + 2.0 × 10⁻² 3a 1.9 × 10⁹ 4.4515 4.06 ± 8.85 × 10⁻² Unavailable 3b 1.9 × 10⁹ 4.45 18 3.84 ± 6.99 ×10⁻² Unavailable 3c 1.9 × 10⁹ 4.45 24 3.42 ± 8.55 × 0⁻² 3.07 ± 1.0 ×10⁻¹ 3d 1.9 × 10⁹ 4.45 36 2.77 + 4.24 × 10^(.2) 2.26 + 1.0 × 10⁻² 3e 1.9× 10⁹ 4.45 72 1.81 ± 5.36 × 10⁻² 0.95 + 5.0 × 10⁻² 3f 1.9 × 10⁹ 4.45 243.12 ± 4.09 × 10⁻² Unavailable 4a 1.9 × 10⁹ 4.45 29 3.07 ± 6.45 × 10⁻²Unavailable 4b 1.9 × 10⁹ 4.45 36 2.76 ± 3.95 × 10⁻² Unavailable 4c 1.9 ×10⁹ 4.45 48 2.25 ± 3.47 × 10⁻² 2.17 + 8.0 × 10⁻² 4d 1.9 × 10⁹ 4.45 721.75 ± 2.03 × 10⁻² 1.61 ± 6.0 × 10^(.2) 4e 1.9 × 10⁹ 4.45 150 0.89 ±1.54 × 10⁻² 0.83 ± 6.0 × 10⁻² 4f 1.9 × 10⁹ 4.45 48 2.24 + 3.49 × 10⁻²2.53 ± 4.7 × 10⁻¹ 5a 1.9 × 10⁹ 2.5 15 Unavailable 1.60 ± 1.90 × 10⁻¹ 5b1.9 × 10⁹ 2.5 15 Unavailable 4.79 ± 3.18 × 10⁻¹ (naphthalene 8.0)

Model predictions and data both demonstrate that salicylate conversionwas limited by reaction rate and not mass transfer effects. Flow rates,effectiveness factors, Thiele modulus, Reynolds numbers, and Biotnumbers are listed in Table 9. Biot numbers were on the order of 100.Effectiveness factor calculations using the best-fit first-orderreaction rate constant indicate that the beads were limited by thereaction rate and not by internal mass transfer resistance. The good fitobtained using the analytical solution, wherein external and internalmass transfer was assumed negligible, also support the conclusion thatmass transfer effects were minimal. The model predicts that thesalicylate distribution reaches a steady state within an alginate bead(99% of final) in about 7 minutes. Even assuming that the diffusioncoefficient was reduced by an order of magnitude in the alginate matrix,a steady state was reached within 15 min. Oyass et al. (1995) found thatdiffusion coefficients in calcium alginate were approximately 85% thatin water for nine solutes (mono- and disaccharides and organic acids).Cell volume estimated at less than 1% probably had little effect ondiffusion. The model also predicted that liquid mass transfer resistancewould have little effect on reaching steady state. In the case of anadsorbing substrate such as naphthalene, the model demonstrates thatadsorption must be taken into account. A positive 50% error in themeasured adsorption coefficient would result in doubling the timerequired to reach steady state. The model predicted that steady statefor naphthalene was achieved in approximately 1 hour starting from aclean alginate bed. Model parameters have been measured (liquid masstransfer coefficient, pellet void volume, and adsorption coefficients),reported, and estimated. Respective coefficients for naphthalene were9.0×10⁻⁶ cm² s⁻¹, 9.0×10⁻⁸ cm² s⁻¹ and 8.4 cm² s mol⁻¹ for liquiddiffusion, solid diffusion, and Langmuir equilibrium constants.Companion free cell studies are needed to further aid evaluation of freeand immobilized kinetics and response. TABLE 9 FLOW RATES, REYNOLDSNUMBERS, THIELE MODULI, EFFECTIVENESS FACTORS, AND BIOT NUMBERS¹Reynolds Effectiveness Study Flow Rate (cm/s) Number Biot Number ThieleModulus Factor 1A 1.17 × 10⁻² 2.65 × 10⁻¹ 1.53 × 10² 9.25 × 10⁻¹ 9.47 ×10⁻¹ 1B 2.92 × 10⁻³ 6.62 × 10⁻² 9.66 × 10¹ 9.25 × 10⁻¹ 9.47 × 10⁻¹ 1C1.17 × 10⁻² 2.65 × 10⁻¹ 1.53 × 10² 9.25 × 10⁻¹ 9.47 × 10⁻¹ 2A 1.17 ×10⁻² 2.65 × 10⁻¹ 1.53 × 10² 9.07 × 10⁻¹ 9.49 × 10⁻¹ 2B 1.05 × 10⁻² 2.38× 10⁻¹ 1.48 × 10² 9.07 × 10⁻¹ 9.49 × 10⁻¹ 2C 2.92 × 10⁻³ 6.62 × 10⁻²9.66 × 10¹ 9.07 × 10⁻¹ 9.49 × 10⁻¹ 3A 1.17 × 10⁻² 2.65 × 10⁻¹ 1.53 × 10²6.77 × 10⁻¹ 9.71 × 10⁻¹ 3B 9.36 × 10⁻³ 2.12 × 10⁻¹ 1.42 × 10² 6.77 ×10⁻¹ 9.71 × 10⁻¹ 3C 7.02 × 10⁻³ 1.59 × 10⁻¹ 1.29 × 10² 6.77 × 10⁻¹ 9.71× 10⁻¹ 3D 7.02 × 10⁻³ 1.59 × 10⁻¹ 1.29 × 10² 6.77 × 10⁻¹ 9.71 × 10⁻¹ 3E4.68 × 10⁻³ 1.06 × 10⁻¹ 1.13 × 10² 6.77 × 10⁻¹ 8.71 × 10⁻¹ 3F 2.34 ×10⁻³ 5.30 × 10⁻² 8.97 × 10¹ 6.77 × 10⁻¹ 9.71 × 10⁻¹ 4A 1.17 × 10⁻² 2.65× 10⁻¹ 1.53 × 10² 6.77 × 10⁻¹ 8.71 × 10⁻¹ 4B 9.36 × 10⁻³ 2.12 × 10⁻¹1.42 × 10² 6.77 × 10⁻¹ 9.71 × 10⁻¹ 4C 7.02 × 10⁻³ 1.59 × 10⁻¹ 1.29 × 10²6.77 × 10⁻¹ 9.71 × 10⁻¹ 4D 7.02 × 10⁻³ 1.59 × 10⁻¹ 1.29 × 10² 6.77 ×10⁻¹ 9.71 × 10⁻¹ 4E 4.68 × 10⁻³ 1.06 × 10⁻¹ 1.13 × 10² 6.77 × 10⁻¹ 9.71× 10⁻¹ 4F 2.34 × 10⁻³ 5.30 × 10⁻² 8.97 × 10¹ 6.77 × 10⁻¹ 9.71 × 10⁻¹¹Flow rates approached creeping flow. Blot numbers ranged around 100,indicating that fluid-phase mass transfer was not significant.Effectiveness factors approached 1.0, indicating that the reaction ratemostly limited the overall reaction rateBioluminescence

Control studies were conducted to determine the effect of glucose, andmineral salts media (no carbon) on the light emission of HK44. Measuredeffluent oxygen levels were always in large excess (>10 ppm).Significant growth of HK44 was improbable as phosphate was not suppliedto the reactor. Glucose is not an inducer for the lux pathway.Bioluminescent activity was constant and minimal using only mineralsalts media. In further studies, glucose in mineral salts media wassupplied to HK44 at 5 mg/L at a flow rate of 1.0 mL/min. When exposed toglucose, light emission was orders of magnitude less than the salicylateresponse but rose slowly for 26 hours above baseline noise. Emission wasthen steady for 12 hours until glucose was removed from the feed.Glucose is an excellent carbon and energy source and could possiblyincrease pools of one or more of the light substrates. Schell (1990)found that low levels of nah mRNA were present in uninduced cells. Thetrizma™ base buffer in the mineral salts feed maintained the pH at 7.2,even for studies with long residence times. Thus, pH does not appear tobe a factor in the light response. These data suggest that the slightincrease in light emission resulted from increased pools of reactionsubstrates and the presence of low, constitutive levels of lux enzymes.

During degradation studies, glucose solution was added to the reactor ata constant concentration throughout the study. Inducer was added 15hours after starting the study. In almost all cases, light intensitymimicked the change in salicylate concentrations. As inducer increased,light intensity increased. Conversely, when inducer concentrations weredecreased, light intensity decreased. The one exception to this behaviorwas observed only at the beginning of a study when a clean bed of HK44was initially shocked by a step change in inducer concentration. Underthis shock condition, light production initially increased orders ofmagnitude and reached a maximum approximately one residence time later(FIG. 25). Within four to six residence times, light intensityapproached a steady state. In contrast, effluent concentrations becameconstant within two residence times. Thus, the unsteady-state lightemission might result from an initial buildup of salicylate within thecell due to the rapid change in the salicylate bulk phase concentrationand an imbalance between transport through the cell membrane anddegradation. Also, addition of the glucose for 15 hours prior toaddition of the inducer may have increased the light substrate pools,resulting in an oversupply of lux cofactors. Thus, further researchwould be appropriate for investigating this transient phenomena. Forexample, cofactor concentrations might be directly measured as afunction of supplied substrates.

FIG. 26 depicts the average specific light response (light per unitbiomass) as a function of predicted salicylate concentration at thelight probe. The data in FIG. 26, obtained during degradation studies,are depicted as averages and standard deviations. During all studies,oxygen was maintained at a level that exceeded the stoichiometricdemand. Oxygen effluent concentrations were always greater than severalmilligrams per liter. The light intensity depicted in FIG. 26 isreported as a function of the local inducer concentration at the lightprobe rather than the effluent concentration. The local salicylateconcentrations were calculated using the model and parameters previouslydiscussed. The purpose of FIG. 26 is to qualitatively compare therelationship between emission and inducer concentration. There was apositive relationship between light emission and inducer concentration.Best-fit lines had slopes of 1.4, 2.3, and 1.5 for studies 2 a-c, 3 c-e,and 4 c-f, respectively. The present analysis must be treatedqualitatively because light values were relative within a study as therewas no reference light source for calibration between studies (e.g.,variable alginate opacity). Thus, the true intercepts for the curves areunknown; however, the slopes were similar and indicate that lightemission was a positive function of inducer concentration.

Studies were conducted to investigate the response to naphthalene (aparent compound of salicylate). Salicylate was added to the PBR as acontrol at 16 hours (study 5 a). The response to salicylate was the sameas in previous studies. Light emission was allowed to become steadyprior to naphthalene addition.

The light response of alginate-immobilized HK44 was intense upon theaddition of naphthalene at 45 hours. The steady-state response of HK44to naphthalene was approximately two orders of magnitudes greater thanthe response to salicylate under identical conditions. These resultswere verified when studies were repeated.

In a second set of studies conducted independently of the degradationstudies, light intensity was measured as a function of inducerconcentration. The degradation studies were not well suited formeasuring light response. In these studies, HK44 was immobilized in athin layer of alginate affixed to a photodiode. A very short residencetime (3 minutes.) was maintained within the flow cells. Further, thesestudies differed from those of the degradation studies in that the cellswere not shocked by a step change in inducer concentration. Rather, thesalicylate concentration was gradually increased. FIGS. 27 and 28 depictthe light response and inducer concentration. Light intensity mimickedthe inducer concentration, although there was a lag. In the naphthalenestudy, a much longer lag was observed than in the salicylate study. Thelag was probably caused, at least in part, by mass transport from thebulk solution to the immobilized cells and, in the case of naphthalene,by adsorption onto the alginate. The differences in lag times may alsobe the result of the different ways that naphthalene and salicylate aretransported in to the cell and consumed. In this set of studies,unsteady-state behavior mimicked the inducer concentration.

The HK44 demonstrated a strong response at part-per-millionconcentrations for both naphthalene and salicylate. PAH concentrationsin soils are typically observed in parts per thousand. Optimal use ofHK44 would be the detection of plume fronts where PAH and degradationproduct concentrations are much reduced. The HK44 was much moresensitive to naphthalene than to salicylate. Uptake mechanisms, energylevels of inducers, and effects of inducer on cell membrane liquiditypossibly contribute to differences in the response to HK44 to thesecompounds. Naphthalene preferentially absorbs to lipids, whichpotentially affects membrane liquidity and may result in increasedaldehyde substrates from lipid synthesis. Furthermore, naphthaleneuptake is probably passive. Because salicylate is a charged ion, uptakemay occur by active transport. Because naphthalene is a greater carbonand energy source than salicylate, naphthalene might increase luxsubstrates resulting in elevated light intensity. NOMENCLATUREC_(biomass) biomass concentration (g cells/dm³) C_(D) _(i) dimensionlessconcentration of component i in bulk phase

dimensionless concentration of component i in the liquid phase insidethe pores of the particle

dimensionless adsorbed solid-phase concentration of component i C₁bulk-feed concentration of component i (mol/dm³) C_(i) ₀ feedconcentration of component i (mol/dm³) C_(P) _(i) pore concentration ofcomponent i (mol/dm³) C_(S) _(i) absorbed solid-phase concentration ofcomponent i (mol/dm³) C_(salicylate) concentration (mol/dm³) D₁dispersion coefficient of component i (dm²/s) D_(P) _(i) porediffusivity of component i (dm²/s) K₁ rate constant (mol/s g cells) K₂rate constant (dm³/s g cells) K_(I) liquid-phase mass transfercoefficient (dm/s) L bed length (dm) N_(P) _(i) rate of mass transfer(mol/s dm³) Pe Peclet number with bed length as the characteristiclength r position in the particle (dm) r₀ particle radius (dm) R₁reaction rate (mol/dm³ s) R_(Salicylate) salicylate reaction rate(mol/dm³ s) S surface area (dm²) t time (s) tr mean residence time inthe bed (s) V interstitial velocity (dm/s) z position in the column (dm)Φ dimensionless position in column φ dimensionless position in theparticle ε bed void volume ε_(p) particle void volume ι dimensionlesstime θ_(S) _(i) dimensionless ratio of absorbed concentration inequilibrium with the maximum feed concentration of component i

Example 4 Deployment of Encapsulated Bioluminescent Bacteria 1NNutrient-Depleted Environments

P. fluorescens HK44 generate blue-green light when exposed tonaphthalene or salicylate. The genes for bioluminescence are located ina plasmid that carries a transcriptional fusion between the promoter ofa salicylate hydroxylase gene, nahG, of a naphthalene-degradationpathway and a promoterless luxCDABE gene cassette of Vibrio fisheri(King et al., 1990). The promoterless lux operon and activity aredescribed elsewhere (Shaw et al., 1988).

The quantity of induced light produced by HK44 cells has been shown tobe proportional to the amount of exposed naphthalene or salicylate(Heitzer et al., 1994). In liquid assays, the cells have been shown todisplay a linear luminescence response with 0.72 μg/l to 3.25 mg/lnaphthalene and 0.4 mg/l to 20 mg/l salicylate (Heitzer et al., 1992).The cells have also been shown useful in bioassays for the detection ofnaphthalene in environmental contaminants. In the demonstration of anoptical on-line biosensor with HK44, immobilized cells also provedapplicable as they emitted a specific luminescence response when exposedto naphthalene in soil slurries, JP-4 jet fuel and leachate ofmanufactured gas plant soil. The information from these studies hassuggested that bioluminescent technology might be used in the assessmentof bioavailability and biodegradation of environmental pollutants thatare significant when endpoints and regulatory standards are determined.

Bioluminescence is an expensive metabolic function as it consumesmolecular O₂, and requires reduced flavin mononucleotide, and thesynthesis of an aldehyde substrate (Hastings et al., 1985). The aldehydemust be regenerated through an ATP- and NADPH-mediated cyclic reactionduring extended emission of light. The physiological burdens raise basicquestions regarding the intrinsic capacity of HK44 and similargenetically engineered strains such as Pseudomonas putida B2 to producestable and specific bioluminescence, upon induction in nutritionallychallenged environments.

Materials and Methods

Bacterial Strains

The bioluminescent bioreporter, Pseudomonas fluorescens HK44 (Germancollection of microorganism) was used in this study (King et al., 1990).HK44 carries the catabolic plasmid pUTK21 (nah⁺, sal⁻, tet⁺), whichcontains a nah-lux transcriptional fusion that allows monitoring ofnaphthalene and salicylate availability and degradation. The lux genescassette, luxCDABE, is transfused to the nahG gene of the sal operon andinhibits the catabolism of salicylate via the plasmid-encoded pathway.The salicylate is, however, degraded by enzymes coded by chromosomalgenes.

Culture Conditions

Strain HK44 was grown in 500-ml conical flasks containing 100 ml yeastextract/peptone/glucose (YEPG) growth medium with 14 mg/l tetracycline.The composition of YEPG is described by Heitzer et al., (1992). Theculture was grown at 27° C. on a shaker.

The organism was grown to exponential phase in YEPG medium (A₅₄₆ 0.8),immobilized in an alginate/SrCl₂ matrix and incubated in groundwater.Simulated groundwater was prepared in the laboratory, from a recipeprovided by the in situ groundwater team at Oak Ridge NationalLaboratory, Oak Ridge, Tenn. This recipe was based on the composition ofvarious groundwater samples analyzed by this team when studyinggroundwater contamination. Simulated groundwater contained (mg/literdistilled water) the following ingredients: CaCl₂ 166, MgCl₂.6H₂O 85,BaCl₂.2H₂O 1.8, SrCl₂.6H₂O 0.6, FeSO₄.7H₂O 25, and KNO₃ 17.Double-strength groundwater was prepared and diluted with the respectivebuffer solutions to yield single-strength groundwater with pH levels 3,4, 5, 6 and 7. The following stocks of buffer solutions were used inadjusting the groundwater to the desired pH. A solution of 0.2 Mpotassium hydrogen phthalate/0.2 M HCl was used to adjust thegroundwater to pH levels 3 and 4, a solution of 0.2 M potassium hydrogenphthalate/0.2 M HCl for pH 7. The groundwater was thoroughly mixed,passed through Whatman filter-paper and sterilized by autoclaving.

Incubation and Induction

Encapsulated HK44 was incubated in groundwater and in 0.1×YEPG medium.The encapsulation was done as described elsewhere (Heitzer et al.,1994). A 500-mg sample of alginate beads, encapsulating HK44, weredispensed into sterile 25-ml vials (Pierce, Ill.) containing 3 mlincubation medium, i.e., groundwater (for nutrient deficiency) and0.1×YEPG (for nutrient surplus). For every type of incubation medium,enough vials were prepared such that a set of triplicate vials could besacrificed for induction and analysis. There were 6 induction days: 1,7, 14, 21, 28 and 35. All vials were incubated at 27° C.

Induction of cells was initiated by adding 1 ml induction solution tothe vials. Light output was measured every 30 minutes from time zero upto 5 hours. For the control, representing the uninduced light response,1 ml distilled water and YEP solution were added to triplicate vialsfrom each type of incubation medium. The light values, if any, wereadjusted as the background light from the light obtained from therespective treatment.

Inducer Solutions

Simple (SS) and complex (CS) inducer solutions were used in thisexperiment. The former consisted of sodium salicylate dissolved indistilled water, the latter consisted of sodium salicylate in YEPsolution. Both solutions provided a final concentration of approximately100 mg/l sodium salicylate. YEP in CS denotes yeast extract andpolypeptone at 0.2 g/l and 2 g/l distilled water respectively.

Light Measurements

Bioluminescence was detected using a photomultiplier tube and measuredin amperes. The light output is presented as nA/cfu.

Population Counts

The numbers of viable-cell-colony-forming units (cfu) of HK44 weredetermined for the encapsulated beads. Encapsulated HK44 were freed bydissolving the alginate matrix with 0.5 M sodium hexametaphosphate,serially diluted in phosphate-buffered saline and spread on YEPG/agarplates containing 14 mg/l tetracycline. The plates were incubated at 27°C. for 36-60 hours and the bacterial colonies were counted.

HPLC Analysis

The concentration of salicylate was determined by high-performanceliquid chromatography (HPLC) before and after the induction response. A2-ml sample of supernatant was withdrawn from each vial of a set foreach treatment type and filtered through 0.2-μm-pore-size Teflonmembrane filters to remove cells and debris of alginate beads prior toHPLC analysis. The HPLC unit consisted of a LC 250 binary pump(Perkin-Elmer, Groton, Conn.) and a Supelcosil LC-18 column (Supelco,Bellefonte, Pa.) and a LS-235 photodiode array detector (Perkin-Elmer).Chromatographic conditions were a continuous gradient from 0 to 60%aqueous acetonitrile between 0.5 minutes and 8 minutes and a secondcontinuous gradient from 60% to 100% acetonitrile between 9 minutes and14 minutes. The program ended with column equilibration for 2.0 minuteswith 100% water. HPLC-grade acetonitrile and water were used in theanalysis. The UV absorbance for salicylate was determined by running a20-μl volume of the sample and detecting the peaks at wavelengths of 255nm. The concentrations were calculated from a standard curve preparedwith known quantities of the sodium salicylate dissolved in high-qualitywater.

Results

Data represent results from one of the three separate repetitions ofthis example. Encapsulated P. fluorescens HK44 responded to inductionwith both SS and CS after incubation in groundwater and 0.1×YEPG. Theresponse time, bioluminescence magnitude and survivability varieddepending on the pH and composition of the inducer solution. Theobservations were made the 5 hours of induction for the 6 differentdays. Throughout the experiment, the pH of the incubation mediumfluctuated within ±0.25 unit.

Induction with SS and CS

The sodium salicylate in the inducer solution induced the lux genes andincreased light emission over time (FIG. 29A and FIG. 29B). Nobioluminescence was observed from cells in groundwater with pH less than6 for either of the inducers. In the other incubation conditions shownin FIG. 29A and FIG. 29B, the logarithmic light levels indicate thespecific and maximum response within the 5-hours post-induction period.The light levels were normalized on the basis of the number of viablecells (cfu) in the alginate/SrCl₂ beads. The light levels were one orderof magnitude higher with CS and than with SS.

As shown for induction by SS in FIG. 29A, log luminescence remainedconsistent in pH 6 groundwater on all days except day 1, the lightmagnitude ranging between 2e⁻⁶ and 9e⁻⁶ nA cfu⁻¹. In pH 7 groundwaterand 0.1×YEPG, a cyclic pattern in the magnitude of the maximum light wasobserved. For instance, the response declined gradually during the firsthalf of the experiment and progressively increased on later inductions(days 28 and 35).

When induced with CS, distinct responses were observed in groundwaterand in 0.1×YEPG. As shown in FIG. 29B, the light levels from cells in pH6 groundwater and at pH 7 were roughly stable on all the induction days.The responses observed in groundwater at pH 6 and 7 were almost similarin pattern when compared to the response in 0.1×YEPG, which steadilydeclined over the days. Nonetheless, with SS or CS, encapsulated HK44indicated a capability for periodic induction for at least 35 days.

The lag time for response may be considered a vital indicator of thephysiological status of the encapsulated cells. This was measured as thetime interval in which the light level increased above the time-zerolevel. In groundwater at pH 6 and 7, the lag time remained the same forthe first 3 induction days and was then extended at pH 7 by about 1 hourwith SS. In 0.1×YEPG, however, it remained the same on all the inductiondays. Interestingly, the lag time was same on all days regardless of theincubation medium with CS.

Salicylate Uptake by Immobilized HK44

The concentration of salicylate before and after induction was used tocalculate the percentage of salicylate uptake. The percentage specificuptake was determined from the number of viable cells (cfu). The dataindicate that salicylate was well in excess during the 5-hour period, asonly 50%-60% of the initial concentration was utilized (FIG. 30A andFIG. 30B). In the presence of SS, the percentage uptake in pH 7groundwater was highly consistent compared to in pH 6 groundwater and0.1×YEPG, indicating the combined effect of pH and starvation onencapsulated cells. With CS, on the other hand, uptake remained almostconstant (approx. 20%), at pH 6 for all inductions except on days 1 and35, displaying a stable response by encapsulated cells. In pH 7groundwater and 0.1×YEPG, a cyclic pattern was observed with a gradualdecline until day 21 and a steady increase on days 28 and 35.

Survivability of Immobilized HK44

The cell viability was determined by plating an aliquot of the dissolvedbead suspension on tetracycline (14 mg/l) containing YEPG/agar medium.The numbers of colony-forming units are shown in FIG. 31. These valueswere stable in 0.1×YEPG, and groundwater at pH 6 and pH 7 during the35-day period. They were highly affected in groundwater with pH below 6and declined below the detection level on day 21. They became detectableon days 28 and 35 for unknown reasons.

Bioluminescence Reaction Rate

Light production was monitored every 30 minutes and the reaction ratewas calculated as nA min⁻¹ cfu⁻¹ for all assay times within the 5-hourspost-induction period. A set of normalized light levels are plotted inFIG. 32 for pH 6, pH 7 groundwaters and 0.1×YEPG. The light levelsindicate a linear increase in luminescence over time in the presence ofsaturating concentrations of salicylate. However, the trend andmagnitude of the rate increase differed, depending on the induction dayand solution. For the two inducer solutions, the rate increase in pH 6groundwater, on all the induction days, was lower with SS than with CS.On day 1, the delayed response may be attributed to non-acclimatizationof cells. In the case pH 7 groundwater and 0.1×YEPG, the response trendand magnitude were comparably similar.

The slopes from the regression fit for the light response are shown inTable 10 for each of the induction events. With SS, except on day 1, theslope in pH 6 groundwater was stable within the same order of magnitude.However, with CS the absolute value of the slope increased in increasingdays of incubation. With SS, the slope values in pH 7 groundwater and0.1×YEPG fluctuated in magnitude. Regardless of the inducers, increasedslope values were observed in groundwater at pH 6 and 7 during the laterstages of the experiment. TABLE 10 RATE OF CHANGE OF BIOLUMINESCENCERESPONSE^(A) Time Incubation Medium Inducer (Days) pH 6 GW pH 7 GW 0.1 ×YEPG SS 1 1.48e⁻⁹ (>0.99) 1.31e⁻⁶ (0.96) 5.78e⁻⁸ (0.88) 7 3.94e⁻⁷ (0.98)5.84e⁻⁸ (0.93) 1.24e⁻⁷ (0.95) 14 8.78e⁻⁷ (0.98) 1.64e⁻⁸ (0.90) 4.58e⁻⁹(0.84) 21  3.2e⁻⁷ (0.97)   9e⁻¹⁰ (0.93) 1.56e⁻⁸ (0.1) 28 5.56e⁻⁷ (0.97)5.68e⁻⁹ (0.90) 1.91e⁻⁷ (0.96) 35 1.01e⁻⁶ (0.98) 1.87e⁻⁷ (0.86) 8.84e⁻⁸(0.70) CS 1 1.49e⁻⁶ (0.96) 5.38e⁻⁶ (0.93) 8.82e⁻⁶ (0.01) 7  9.9e⁻⁷(0.96) 2.48e⁻⁷ (0.98) 1.75e⁻⁷ (0.80) 14 1.55e⁻⁶ (0.95) 3.44e⁻⁷ (0.96)6.51e⁻⁸ (0.88) 21 1.56e⁻⁶ (0.91) 7.74e⁻⁸ (0.98)  1.7e⁻⁸ (0.37) 283.04e⁻⁶ (0.95) 1.87e⁻⁷ (0.94) 1.45e⁻⁷ (0.05) 35 4.52e⁻⁶ (0.92) 6.98e⁻⁷(0.95) 9.33e⁻⁸ (0.09)^(A)The values refer to the slope of a linear curve fit for the lightresponse observed within the 5-hour post-induction period. The r2 of thelinear fit is given in parenthesis.GW groundwater;YEPG yeast extract/peptone/glucose medium;inducer solutions;SS simple solution,CS complex solution.Discussion

Observations made in this example supported evidence for a frequentresponse upon induction, measurable light emission and survival of theencapsulated P. fluorescens HK44 under nutrient-limiting conditions. Inaddition, the encapsulation process by itself proved sustainable forlong-term biological activities. These features are critical in thedesign and application of a field biosensor using P. fluorescens HK44.

Among the simulated environmental conditions tested in this study, thecells distinctly preferred pH 6 and 7 groundwater for efficientinduction and survivability. This reflected the potential limitation inthe direct application of HK44 since they failed to respond ingroundwater with pH below 6, either because of inhibition of thebioluminescence reaction or of cell viability or both. Interestingly, inmany naturally bioluminescent bacteria the optimal pH for luciferaseactivity is reportedly slightly acidic (Danilov and Ismailov, 1989).

Concerns regarding the continuous effectiveness of the bacteria in along-term biosensor application were cautiously addressed in this study.As observed, the bioluminescence reaction rate differed in magnitude andtrend, in groundwater at pH 6 and pH 7 over the 35-day period. If a“cut-off” performance period can be derived for each of the groundwatersamples; the performance efficacy of the bacteria can be set for adefinitive time frame, allowing replacement of the old encapsulatedcells with new at the end of the time frame and rendering the biosensorcapable of continuous operation. For instance, in the present study, aconservative cut-off period of 28 and 14 days might be set for pH 6 and7 GW respectively on the basis of the response (Table 10, FIG. 29A andFIG. 29B).

Encapsulation proved supportive for this type of long-term application.However, there was no indication that it influenced substrate intake orthe bioluminescence reaction on the basis of studies conducted with freecells. Similar comparisons in Pseudomonas sp. also have revealed thatimmobilization has no generalized effect on the physiological activity(Shreve and Vogel, 1993).

Example 5 Immobilization and Encapsulation of Microbial Cells onIntegrated Circuits

The deposition of microbial organisms on integrated circuits may beaccomplished through the various protocols described below. The ultimategoal of these encapsulation methods is to provide the cells with astable microenvironment limited from the stresses of their outerenvironment. Encapsulated cells can be formed into sheets or beads,almost of any thickness or diameter desired, depending on the methodchosen. The small area available for cell deposition on an integratedcircuit requires thin sheets (0.1-2 mm) or small diameter beads (<50 μm)to be produced. However, the high sensitivity of the integrated circuitallows for a smaller cell mass to be used. For the procedures below acell culture containing about 1×10⁶ to about 1×10⁸ cfu/ml is typicallygrown and an about 1 to about 5 g wet weight of these cells may beutilized in the encapsulation protocol.

Numerous matrices are available for encapsulation. Polydimethylsiloxane(PDMS) is a silicone elastomer that molds and adheres to an integratedcircuit surface. Polyvinyl alcohol/polyvinyl pyridine (PVA/PVP)copolymer is a biocompatible material suitable that has been used forenzyme immobilization and adheres well to graphite electrode surfaces.Latex copolymers may be utilized in various ways; for example,immobilization of cells in a porous vinyl acetate lower layer sealedwith an upper layer. Cells may in some instances be adhered to a surfaceby electrophoretic deposition using low current densities.

Agar/Agarose

Cells may be added to molten agar or agarose (from about 1% to about5%). Gelation occurs as the agar or agarose cools to room temperature(Kanasawud et al., 1989).

Carrageenan

A 2% solution of carrageenan may be warmed to about 70° C. to about 80°C. to initiate dissolution and then maintained at a temperature of fromabout 25° C. to about 50° C. The cell culture also is warmed and addedto the carrageenan solution. Gel formation occurs through the additionof cold 0.1 M potassium chloride.

Polyacrylamide

Cells are mixed in a solution of acrylamide (35 g) and BIS (2.4 g).Ammonium persulfate (40 μl of a 0.40 g/ml solution) and TEMED (100 μl)are then added to initiate polymerization. Within 20 minutes sheets ofencapsulated cells of any desired thickness can be sliced. Cell dropletsmay also be added through a syringe to the acrylamide solution toproduce beads of encapsulated cells (of from about 1 mm to about 3 mm indiameter). Small diameter microbeads (from about 2 μm to about 50 μm)may be produced by spraying the cell mixture through a nebulizer orvaporizer.

Alginate

Cells are added to an about 1% to about 8% solution of alginate.Addition of 0.5 M calcium chloride or 0.1 M strontium chloride initiatespolymerization. Sheets, beads, or microbeads may be produced.

Alginate encapsulated cells may be encased in 0.1 μm low adsorptionfilter membranes and hollow fiber membranes to allow inflow of analytes.This will inhibit alginate degradation and cellular release into thesurrounding medium.

Polyurethane/Polycarbomyl Sulfonate (PCS)

Polyurethane or PCS at a polymer content of 30-50% is mixed with a 1%calcium chloride solution. The pH is adjusted to approximately 6.5 andthe cell mass is added. This mixture is sprayed into 0.75% calciumalginate and beads are formed. After one hour the beads are removed,washed, and introduced into a 2% sodium tripolyphosphate buffer whichdissolves the alginate layer leaving only a layer of polyurethane/PCSsurrounding the cells.

Polyvinyl Alcohol (PVA)

The cell suspension is mixed with a 13% PVA, 0.02% sodium alginatemixture. Upon contact with a solution of saturated boric acid and 2%calcium chloride, gelation occurs.

Sol-Gel

The sol-gel process allows for the formation of silicon glass under roomtemperature conditions. Cells are combined with 0.1 M Tris-Cl andtetramethylorthosilicate (TMOS), tetraethylorthosilicate (TEOS),methyltrimethoxysilane (MTMS), ethyltrimethoxysilane (ETMS),propyltrimethoxysilane (PTMS), or polydimethylsiloxane (PDMS).Solidification times vary depending on the concentrations used (of fromabout 0.02% to about 0.5%). Sheets, beads, or microbeads can be produced(Armon et al., 1996).

Combination of Procedures

Many of the above methods can be combined. For example, cells can firstbe encapsulated in alginate, carrageenan, agar, or agarose and thenencapsulated again in a stronger layer of PCS, PVA, or sol-gel. Layersof encapsulation can also be produced; alginate microbeads can be‘sandwiched’ between layers of sol-gel. This provides the cell with agreater degree of protection than a single layer alone and allows theouter layer to be more compatible with the integrated circuit whilemaintaining an inner layer more compatible with the cells.

Amendments

Various amendments can be added during the encapsulation process to aidin cell survival. These include oxygen carriers such aspolydimethylsiloxane; nutrient sources such as powdered skim milk;moisture reservoirs such as clay particles; and compounds to improvestrength and flexibility, such as bean gum.

Example 6 Toxicity Applications of Bioluminescence

A number of assays have been developed that allow the measurement oftoxicity of a given compound or compounds based on the effect of thecompound or compounds on bioluminescent bacteria. Basically, toxicity isindicated by a decrease in the bioluminescent signal of the testbacteria. Commercially available assays include the Microtox and theLumitox systems. These assay systems utilize bacteria that are naturallybioluminescent. Examples of applications of toxicity assays usingbioluminescent bacteria are given in Table 11, including the type oforganism used and the name of the assay, if commercially available.

Example 7 Bioluminescent Genotoxicity Assays

Recently, a number of assays utilizing bioluminescent bacteria have beendeveloped to determine whether a compound is a mutagen or whether amutagenic compound has contacted the bacteria. The assays are based onthe ability of a suspected mutagen to cause distinct changes in thebacterial DNA allowing bioluminescence or the response of cells todamaged DNA caused by the mutagen. Examples of applications ofbioluminescent genotoxicity assays are given in Table 12, including thetype of organism and bioluminescence genes used.

Example 8 Methods of Screening Antimicrobial Agents

Organisms that are naturally bioluminescent or that have been engineeredto be bioluminescent may be used to screen compounds for their abilityto affect the viability of the organism. Basically, in these assays,bioluminescence will be inversely proportional to biocidal activity.Examples of applications of bioluminescent antimicrobial screeningassays are given in Table 13, including the type of organism used andbioluminescence genes (if applicable). TABLE 11 TOXICITY APPLICATIONS OFBIOLUMINESCENCE TEST ORGANISM LUX GENES ASSAY APPLICATION P. phosphoreumN.A.¹ Microtox System may be used to screen whether or not tributenyltincompounds were less toxic than tributyltin compounds as an antifoulingagent. P. phosphoreum Microtox System may be used to determine whetheror not pesticides in the soil were more or less toxic than the productsof their degradation. P. phosphoreum Microtox Assay may be used todetermine the distribution of pollution in the sediment interstitialwaters of the Detroit River. P. phosphoreum Microtox Assay may be usedto determine the toxicity of breakdown products from phenolic compoundsas they apply to waste water treatment. P. phosphoreum Microtox Assaymay be used to determine the toxicity of Trinitrotoluene,Diaminotoluene, and Dinitromethylaniline mixtures. P. phosphoreumLumitox Assay may be used to examine the discharges into the RiverTormes in Salmanca Spain and correlate the decrease in bioluminescenceto the impact on the river. P. phosphoreum Microtox Assay may be used toexamine the usefulness of bioluminescence to detect cyanobacterialblooms and the associated hepatotoxins (microcystins). V. harveyi NAAssay may be used to detect biohazardous chemicals in soil and waterextractions with and without acid.. V. harveyi NA¹ Assay may be used toevaluate combined or mixture toxicity of two organic compounds,nitrobenzene and trinitrobenzene. V. fischeri Microtox Assay may be usedto determine the impact of point and nonpoint pollution on pore watersof two Chesapeake Bay tributaries. V. fischeri Microtox Assay may beused to determine the effect of river and wetland sediments on thetoxicity of metolachlor. P. phosphoreum Microtox Assay may be used todetermine petroleum hydrocarbon toxicity. P. phosphoreum Microtox Assaymay be used to determine the efficacy of ultrafiltration for removal oforganics from groundwater. P. phosphoreum Microtox Assay may be used totest the toxicity of marine surfactants. P. phosphoreum Microtox Assaymay be used to determine the acute toxicity of Euphorbia splendenslatex. P. phosphoreum Microtox System may be used to test for thepresence of paralytic shellfish poison-like neurotoxins in a “red tide”bloom of Gonyaulax polyedra. P. phosphoreum Microtox Assay may be usedto determine the toxicity of thio- and alkylphenols causing flavortainting of fish from the upper Wisconsin River. P. phosphoreum MicrotoxAssay may be used to test the toxicity of ozonolysis by-products indrinking water P. phosphoreum Microtox Assay may be used to determinebiological effects of certain metals and organic compounds found in woodpreservatives. P. phosphoreum Microtox Assay may be used to determinethe toxicity of 4- chloro-2-methylphenoxyacetic acid P. phosphoreumMicrotox Assay may be used to determine the toxicity of water samplesand extracts from the Sora river area. P. phosphoreum Microtox Assay maybe used to determine the toxicity of Lake Orta (Northern Italy)sediments. P. phosphoreum Microtox Assay may be used to determine thelethal effects of azulene and longifolene. P. phosphoreum Microtox Assaymay be used to determine the toxicity of granular activated carbontreated coal gasification water. P. phosphoreum Microtox Assay may beused to assess copper complexation with organic compounds.¹NA, not applicable.

TABLE 12 GENOTOXICITY ASSAYS TEST ORGANISM LUX GENES APPLICATIONPhotobacterium NA¹ A dark variant of P. phosphoreum is marketed fromphosphoreum (dark Micrabic Corporation under the name Mutatox. Thisvariant) system monitors genotoxicity by exposing the bacteria to thesuspected mutagen and if reversion to bioluminescence occurs it suggeststhe compound is a possible mutagen. E. coli Firefly luciferase insertedPhage λ is integrated into the chromosome of E. coli into phage λ toexpress the forming a lysogenic strain. Since mutagens have theluciferase in the prophage ability to induce prophage λ bioluminescencewould form indicate the presence of a suspected mutagen. Since thisassay uses the luciferase only luciferin has to be added exogenously. E.coli luxAB of V. fischerii inserted Assay is the same as above exceptthe substrate for the into phage λ to express the luciferase isn-decanal. luciferase in the prophase form¹NA, not applicable.

TABLE 13 SCREENING ANTIMICROBIAL AGENTS TEST ORGANISM LUX GENESAPPLICATION Mycobacterium Firefly luciferase cloned in Strain used toascertain the effectiveness of various tuberculosis front of heat shockpromoter antimicrobial agents. Assay is performed in vitro as the on theshuttle vector cells were lysed and the luciferin substrate added.pMV261 However, luciferin can be added to whole cells. MycobacteriumluxAB from V. harveyi cloned Strain used as a rapid way to screen foreffectiveness of. smegmatis in front of heat shock antimicrobial agents.Assay uses whole cells, but promoter in the shuttle vector requires theaddition of the aldehyde substrate. pMV261 Listeria monocytogenes luxABfrom V. fischerii Assay used to evaluate the effectiveness of peroxygencloned in an expression disinfectant as a biocide for the intracellularpathogen L. monocytogenes. plasmid Listeria monocytogenes luxAB from V.fischerii Assay used to examine the biocidal effect of phenol and clonedin an expression chlorohexidine on the intracellular pathogen L.monocytogenes. plasmid Photobacterium NA¹ Assay used to ascertain theeffectiveness of using phosphoreum acoustic energy and cavitation onbacteria by examining bioluminescent levels while varying acousticpressures and duration. One application is the inhibition ofcolonization of the oral cavity. E. coli and B. subtilis luciferase genefrom Assay may be used to determine the membranolytic pyrophorusactivity of serum complement. plagiophthalamus¹NA, not applicable.

Example 9 Pollution Detection Using Bioluminescence Assays

Common features of microbial metabolism include the ability to recognizea compound in the environment, turn on the expression of genes requiredto utilize the metabolite, and, subsequently, turn off these genes whenthe metabolite is no longer present. the classic example is the lacoperon. The lac operon promoter is repressed in the presence of simplesugars or the absence of lactose. However, when simple sugars are notavailable and lactose is present, the lac operon is highly expressed.When the level of simple sugars is sufficient or the lactose isdepleted, the lac operon again is repressed.

Microorganisms have the ability to metabolize a wide variety ofcompounds. Some bacteria are able to metabolize compounds that are toxicto humans and are considered pollutants. Expression of the genes thatenable pollutant metabolism is similar to that of the lac operon.Certain bacteria can recognize the presence of the pollutant in theenvironment, turn on the genes required for metabolism of the pollutant,and repress the genes when the pollutant is no longer present. Byoperatively linking a gene or genes that provide bioluminescence to apromoter of a pollutant metabolism gene or operon, one may detect themicroorganism's response to the presence of the pollutant. Severalexamples of such a utility are given in Table 14, including theorganism, lux genes, and the promoter to which the lux genes areoperatively linked.

Example 10 Bioluminescent Oxygen Sensor

The ability of Photobacterium to emit light in response to molecularoxygen has been used to monitor low dissolved oxygen concentrations(Lloyd et al., 1981). Other examples are given in Table 15. TABLE 14POLLUTANT DETECTION: AROMATIC COMPOUNDS AND STRESS INDICATORS TESTORGANISM LUX GENES APPLICATION P. fluorescens HK44 nah-luxCDABE (V.fischerii) Strain able to semiquantitatively determine naphthaleneconcentrations. Also used in an on-line optical biosensor to determinethe presence of naphthalene in water flowing past the sensor. P. putidaB2 tod promoter cloned in front of Strain detects toluene in watersamples as well as the promoterless lux genes of water-solublecomponents of JP4 jet fuel. Strain used in pUCD615 the on-linemonitoring of TCE degradation in a differential volume bioreactor. E.coli two heat shock promoters Strains treated with a variety ofenvironmental insults dnaK and grpE were fused to including ethanol andpentachlorophenol; showed an V. fischerii luxCDABE increase inbioluminescence correlating with the pUCD615 induction of the heat shockresponse. E. coli heat shock promoter grpE E. coli strain harboringgrpE-lux fusion assayed for its fused to the V. fischerii use in aminiature bioreactor to act as an Early Warning luxCDABE pUCD615. Systemfor the detection of toxic levels of pollutants in the influent of awaste water biotreatment plant. E. coli mercury resistance operonBiosensor for the semiquantitiative detection of fused to promoterlessV. fischerii bioavailable inorganic mercury in contaminated waters.luxCDABE E. coli lux operon from Photorhabdus Assay may be used todetect the presence of nitrate. luminescens fused to the nitratereductase (narG) promoter¹N.A. = Not applicable

Example 11 Bioluminescence in Eukaryotic Reporters

The luciferase and green fluorescent proteins have been used extensivelyas reporter genes in Eukaryotic systems. Examples of the use ofluciferase genes in mammalian cell lines are given in Table 16,including the name of the cell line used, promoter and bioluminescencegene used, and a brief description of the application.

Example 12 Measurement of a Bioluminescent Signal by an OASIC

Pseudomonas fluorescens HK44 generate blue-green light when exposed tonaphthalene or salicylate. The genes for bioluminescence are located ina plasmid that carries a transcriptional fusion between the promoter ofa salicylate hydroxylase gene, nahG, of a naphthalene-degradationpathway and a promoterless luxCDABE gene cassette of Vibrio fisheri(King et al., 1990). The promoterless lux operon and activity aredescribed elsewhere (Shaw et al., 1988).

A microscope slide with a culture of Pseudomonas fluorescens HK44 wasplaced over an OASIC. The resulting device was exposed to naphthaleneand the output voltage was measured over time (FIG. 2).

Example 13 Ammonia Biosensor

A DNA fragment containing the promoter region of the sequencedhydroxylamine oxidoreductase gene (hao) (Sayavedra-Soto, et al., 1994)was obtained by PCR amplification using Nitrosomonas europaea ATCC19178chromosomal DNA as template. The amplified fragment was cloned, and thenucleotide sequence of the promoter was confirmed by sequencing. The haopromoter was cloned in front of the promoterless luxCDABE genes fromVibrio fischeri in a mini-Tn5 artificial transposon, which contains akanamycin resistance gene for positive selection of transposition (FIG.49). The transposon delivery vector was introduced into N. europaea bymating between E. coli SV17/pUTK220 and N. europaea ATCC19178 cells, andkanamycin resistance was used to select those clones with the hao-luxfusion in the chromosome of N. europaea (N. europaea Km^(r) hao-lux).Slot blot analysis of eight Km^(r) clones using lux as a probe indicatedthat the clones amo1 and hao3 contained the transposon in the genomicDNA (FIG. 50).

Specific bioluminescence of the hao-lux fusion was measured during thegrowth of N. europaea Km^(r) hao-lux in minimal media in the presence of50 mM (NH₄)₂SO₄ (Ensign, et al., 1993). The culture was shaken at 100rpm at 30° C. in the dark, and sampled for approximately 80 h. Specificbioluminescence (photons/sec/OD) of N. europaea Km^(r) hao-lux cellsshowed an increase with time during the first half of the exponentialgrowth, suggesting that luciferase is accumulating in this time frame(FIG. 51A and FIG. 51B). The specific bioluminescence values steadilydecreased at the end of the exponential phase, and presented backgroundlevels after 150 h of growth (FIG. 52A and FIG. 52B).

To determine the bioluminescence response of the N. europaea hao-luxfusion to increasing concentrations of NH₄ ⁺ , N. europaea Km^(r)hao-lux cells were grown for 150 h in minimal media with 50 mM(NH₄)2SO4, washed in the same media in the absence of NH₄ ⁺, and exposedto 0 to 5 mM (NH₄)₂SO₄ for 30, 60 and 90 min. An increase inbioluminescence was observed with increasing NH₄ ⁺ concentrations at alltested times, being the response saturated at the higher concentrations(FIG. 53). The NH₄ ⁺ detection limits were determined to be 20 μM at 30min of exposure and 10 μM at 60 min or 90 min of exposure (threefoldincrease over background bioluminescence).

Example 14 Estrogen Biosensor

A reagentless yeast bioluminescent reporter system for estrogens andxenoestrogens was constructed by fusing the luxAB genes and the luxCDEgenes from Xenorhabdus luminescens. These fusions allow the expressionof these bacterial genes in eukaryotic cell lines. The modified genecassettes can be expressed in Saccharomyces cerevisiae cells using theyeast expression vector pYES2.

Initially, the luxAB genes were fused by constructing oligonucleotidesthat amplified (using pfu polymerase) both the luxA and the luxB withthe necessary genetic modifications. The resultant fragments were thenbe blunt end ligated and reamplified with the luxA forward primer andthe luxB reverse primer. This approach was unsuccessful, as there werenumerous errant PCR products.

An alternate strategy was to fuse the two genes using an oligonucleotidewhich was complimentary to the 3′-end of the luxA and spanning theintergenic region between the luxA and luxB including the start codon ofluxB. The oligo and its complement were synthesized with the followingmodifications: the luxA stop codon TAG was replaced with the codon fortyrosine by substituting a C for the G; since the stop codon waseliminated a G was also inserted to put the luxA and luxB genes in thesame reading frame; an AvrII restriction site was also placed into theoligonucleotide for fusing the resultant amplified luxA and luxBfragments. The luxA and luxB genes were successfully amplified andcloned into pCRII (Invitrogen, Carlsbad, Calif.). The luxB gene was thencloned into the unique AvrII site in pCRII containing the modified luxAusing the added restriction site. The resultant ligation was subjectedto the polymerase chain reaction using a luxA forward primer and a luxBreverse primer. The resultant PCR fragments were TA cloned and screenedfor light production with the addition of n-decanal. Bioluminescentcolonies were isolated. Restriction analysis was performed on theresultant plasmid constructs to verify the luxAB fusion using theintroduced AvrII site. The fusion showed a similar bioluminescence levelto an unfused luxAB gene cassette.

These results show that the fusion of the two genes to form a monomericprotein did not significantly affect bioluminescence. To facilitate highlevels of expression in the mammalian cell lines it was necessary tomodify the bacterial ribosomal binding site by replacing certain basesto generate a eukaryotic ribosomal binding site. This was accomplishedby mutating the sequence surrounding the luxA initiation codon to a goodKozak context using site directed mutagenesis as previously described byinserting an A at the −3 position and a G at the +4 position. Sinceconstructs were to be screened initially in E. coli for lightproduction, the unfused luxAB cassette with the Kozak modification wasexamined to ascertain whether or not effective translation wasoccurring.

Despite the Kozak modifications, the luxAB in E. coli was poorlyexpressed. These data indicated that the resultant clones of the Kozakmodified luxAB fusion would have to be screened by restriction analysisas well as the absence of light. The Kozak modified luxAB fusion hasbeen successfully amplified and inserted into the galactose inducibleyeast expression vector pYES2 and inserted into S. cerevisiae. Fivesuccessful transformants harboring the galactose inducible luxAB fusionwere evaluated for upregulation of bioluminescence. All five werecomparable. The appropriate fusions have been constructed and will beintegrated with the estrogen response element to provide abioluminescent estrogen bioreporter.

Example 15 Bioreporter Encapsulation

Sol-gel encapsulation studies substituting another yeast strain, S.cerevisiae HER, a β-galactosidase bioreporter for estrogenic activitywere conducted. Strain HER was successfully incorporated into thesol-gel matrix while retaining responsiveness to the estrogen inducerβ-estradiol. As shown in FIG. 54, an estrogenic response was initiatedin encapsulated yeast cells over a seven day period. This responseremained comparable to that of the positive control non-encapsulatedyeast cells.

Polydimethylsiloxane (PDMS) is also useful for encapsulation. PDMS is asilicone elastomer that molds and adheres to the integrated circuitsurface. The lux bioreporter Pseudomonas fluorescens HK44 wasencapsulated in PDMS to generate a bioluminescent response when exposedto the chemical inducer naphthalene. TABLE 15 OXYGEN SENSORS TEST LUXORGANISM GENES APPLICATION P. phosphoreum N.A.¹ P. phosphoreum is usedin this assay as sensor for bacterial oxygen demand (BOD). BOD isdetermined by the increase in bioluminescence. As the organic moleculesin the test water are metabolized reduced products are shunted to thebioluminescence reactions causing an increase in bioluminescence. P.phosphoreum N.A.¹ P. phosphoreum is used in this assay as an onlinecontroller of oxygen concentration. The bacterial oxygen sensor was usedto control the optimal dissolved oxygen concentration to produce maximumC₂H₂ reducing activity in Klebsiella pneumoniae.¹N.A. = Not applicable

TABLE 16 EUKARYOTIC REPORTERS TEST CELLS LUX GENES APPLICATION humanliver cancer cell CYP1A1-luc (firefly) gene A construct engineered suchthat when a toxic line fusion compound which would elicit a P450response it expresses the firefly luciferase instead. The present methodinvolves the lysis of the cells as well as the addition of exogenousluciferin to measure activity however a whole cell assay may bedeveloped. human hepatoma cell line epo promoter sequence fusedbioluminescence used to monitor the induction of the Hep3B to lucerythropoiten gene. Hypoxia found to cause a 4-fold induction of geneexpression in Hep3B. HeLa cells luciferase was fused to a HeLa cellscotransfected with the expression vector thymidine kinase promoter HEG0and the luciferase reporter plasmid harboring a Vit. A2 ERE.Antiestrogens designed and tested and found to inhibit transcriptionalactivity. mouse fibroblast 3T3 cells ribonucleotide reductase Reporterconstructs utilizing the R1 and R2 promoter- promoters for both subunitsluciferase constructs transformed into mouse fibroblast R1 and R2 fusedwith cells. R1 luc shows a 3-fold induction and R2 luc a 10- luciferasefold increase upon exposure to UV light in a dose dependent manner.estrogen receptor-positive reporter plasmid contains a luciferasereporter used to screen for both estrogenicity breast cancer cell linethymidine kinase promoter and antiestrogenicity fused to a fireflyluciferase Hela cells firefly luciferase controlled Chimeric proteinscomprising the DNA binding domain by the Gal-4 promoter of Gal4 yeastproteins and hormone binding domains of various steroid receptors areplaced into the cell lines containing the Gal-4-luciferase construct totest the biological activities of steroid hormones.

Example 16 Bioluminescence Detection

Bioluminescence was determined for cultures containing differentconcentrations of P. fluorescens 5RL cells growing in LB supplementedwith 10 ppm of the inducer molecule salicylate and 14.7 mg/Ltetracycline (FIG. 46). Bioluminescence was determined using theintegrated circuit microluminometer and a light-tight enclosure mountedabove the chip. Linear regression analysis showed that the data fit alinear model indicating that bioluminescence per cell remains constantfor cell concentration ranging from 4× to 2×10⁸ CFU/mL and for detectorresponses ranging from 0.05 to 20 pA. Using a linear model, the limit ofdetection (2 sigma) for this experimental geometry was estimated to be4×10⁵ cells per mL. At cell concentration greater than 4×10⁸ CFU/mL, thebioluminescence decreased possibly due to oxygen limitation caused bythe quiescent conditions of the vial (FIG. 47).

The results obtained with the BBIC microluminometer were compared withresults collected with the Azur luminometer at each cell concentration(FIG. 48). The data showed that the measured bioluminescence responseswere proportional for cell concentrations ranging 4×10⁵ to 2×10⁸ CFU/mL,indicating that the BBIC microluminometer gave consistent resultscompared to standard PMT-based detection systems.

The attractive attributes of the sensor developments described here arethe potentials for creating wholly self contained biosensors thatrequire no exogenous reagents beyond what can be provided on the IC andthat the IC can function independently of any other instrumentalcomponents.

Example 17 Modifications to CFC Circuit for Low Reverse Bias Operation

FIG. 43 shows the portion of the CFC circuit involved in the biasing ofthe photodiode. The switch across capacitor Cf is realized with atransistor. As shown in this figure, a single transistor is usuallyemployed. However, if the leakage current through this transistorexceeds the leakage current of the photodiode, then the circuit does notoperate correctly. In practice this places a lower limit to the reversebias on the photodiode.

FIG. 44 illustrates a solution to this problem. A two-transistor switchwith a path to ground at the central point was employed. Although thepath is shown as a current source, in practice it can be a resistor oranother transistor with a fixed gate voltage. In this circuit, theleakage current passes through the first transistor, but is shunted toground by the current source. Therefore, no leakage current finds itsway to the photodiode node.

Integrated Microluminometer

Microluminometer Chip

FIG. 45 shows a photograph of the complete microluminometer chip. Thechip measures 1.9 mm×1.9 mm with the photodetector occupying ˜33% (1.2mm²) of the total chip area. For testing purposes the chip was mountedin a 40-pin ceramic dual inline package

The level of light that is detectable on the chip is an importantperformance parameter of a BBIC. Modifications to the design of thephotodetector and the front-end analog processing circuitry improved theminimum detectable signal by at least 20-30%. In particular, themodifications include:

-   -   (1) Selection of an n-well/p-substrate junction for        photodetection. This junction has significantly lower noise and        higher quantum efficiency.    -   (2) Selection of an electrode configuration that minimizes        photodiode leakage current and capacitance (both of which        increase noise) without significantly impacting quantum        efficiency.

Additionally, changes have been made to the front-end processingcircuitry which allow the photodiode to work at a reverse bias of 0V.

The many potential applications for BBICs include environmentalmonitoring, food and water quality testing, in vivo sensors for diseasedetection and management, and other remote applications where size,power consumption, and cable plant concerns are the dominant issues.Therefore, the integrated circuit (IC) portion of the BBIC should resideon a single chip, be compatible with battery operation, and becompatible with RF circuits for wireless telemetry in addition toallowing the integration of high-quality photodiodes and low-noiseanalog signal processing. We chose a standard 0.5-μm bulk CMOS processthat meets optical and signal processing requirements, while allowingthe integration of RF circuits operating in the 916-MHz band. The designand performance of the two major components of the microluminometer: theCMOS photodiodes and the front-end signal processing are discussed.

CMOS Photodiodes

CMOS technology allows the realization of phototransistors, photodiodes,and photogates without any modification or additions to the standardprocessing steps. As normally used, these devices have broad spectralresponses that peak in the red/near infrared region. Peak externalquantum efficiency of 50%-80% has been reported for CMOS photodiodes(Kramer, et al., 1992).

FIG. 38 shows two junctions available for the realization of CMOSphotodiodes. The shallower junction (p-diffusion/n-well) is desirablefor this application since its response peaks near the 490-nm wavelengthof the bioluminescence, yet drops off quickly at longer wavelengths(Simpson, et al., 1998). However, the quantum efficiency ofp-diffusion/n-well photodiodes in small geometry CMOS processes is low(typically less than 10%). One possible explanation is that the shorterdrive-in diffusion step for small geometry processes is insufficient toanneal the lattice damage created by the ion implantation step, therebyleaving a high density of charge traps in these diffusions. In thiscase, the large number of charge carrier traps will severely degrade thequantum efficiency in the blue and green optical regimes. Regardless ofthe mechanism, the p-diffusion/n-well junction is not suitable forlow-level luminescence detection, leading to selection of then-well/substrate photodiode for the microluminometer transducer.

The physical layout of the electrodes affects both the quantumefficiency and the reverse leakage current of the photodiode. Twopossible electrode configurations are shown in FIG. 39. In the firstconfiguration the n-well electrode covers the entire active region ofthe photodiode. The advantage of this approach is that all thephoto-generated charge is produced in the n-well and must only diffuse ashort distance to the n-well/substrate junction without being trapped toproduce a photocurrent.

The second approach in FIG. 39 uses an array of small n-well/substratejunctions spread across the active region of the detector. This approachminimizes the degradation of noise performance caused by detectorcapacitance and leakage current. However, charge created in thesubstrate regions must diffuse a relatively long distance without beingtrapped to produce a photocurrent. In principle one could calculate theoptimum spacing between electrodes given a detailed knowledge ofmaterial parameters such as the diffusion length and the surfacerecombination velocity. As these parameters are likely to vary from runto run, empirical determination of optimum spacing may be the beststrategy. For this design, 5.6 μm×5.6 μM electrodes spaced 12.6 μm apartas shown in FIG. 40 were selected.

For use with bioluminescent bioreporters, it is desirable to minimizethe photodiode reverse leakage current for two reasons. First, the powerspectral density of the detector white noise depends directly on themagnitude of the dc leakage current. Possibly more important is theinability to distinguish a low-level dc luminescent signal from a dcleakage current. Variations in the leakage current as a function oftemperature cannot be distinguished from a change in thebioluminescence. Conventional solutions, such as chopping the opticalsignal, are not practical for this integrated, single-chip, analyticalinstrument.

The ideal diode equation, $\begin{matrix}{I_{f} = {I_{s}\left( {{\mathbb{e}}^{\frac{V_{f}}{V_{T}}} - 1} \right)}} & (36)\end{matrix}$where

-   -   I_(f)=forward current    -   I_(s)=reverse saturation current    -   V_(f)=forward bias    -   V_(T)=thermal voltage (≈26 mV@room temperature)    -   describes two competing current components: 1) electrons/holes        on the n/p side overcoming the potential barrier; and 2)        holes/electrons on the n/p side diffusing to the edge of the        space charge region and being swept across        I_(r)≡−I_(s).  (37)        At zero bias these two components are in dc equilibrium, so the        dc leakage current is zero. However, these currents are        uncorrelated, so their noise power spectral densities (PSD) add.        This simple analysis predicts that the noise PSD at zero bias is        higher than it is at any reverse bias.

Unfortunately, the situation is not that simple. Equation (36) describesmoderate to strong forward bias current well. However, at weak forwardbias or in reverse bias, equation (36) under predicts the magnitude ofthe current because of surface and generation/recombination effects.I_(r) as well as I_(f) will depend on bias, and it is not certain atwhat bias level the minimum noise is found. However, zero bias iscertainly where the minimum dc leakage current, and therefore thegreatest immunity from thermally generated false signals, is found.

FIG. 41 shows the reverse leakage current versus reverse bias for thephotodiode of FIG. 40 at three different temperatures. This figureclearly shows that operating at reduced bias greatly reduces themagnitude of the temperature drift of the leakage current. FIG. 42 showsthe measured photodiode signal versus reverse bias for the photodetectorshown in FIG. 40 at four different light levels. This figuredemonstrates that the quantum efficiency has a weak dependence on biasfor reverse biases above 50 mV. In addition, this figure shows thequantum efficiency of this detector to be ˜68% at 490 nm (1.75 pAphotocurrent for an input flux of 1.6×10⁷ photons/sec.), which indicatesthat the spacing between n-well electrodes can be increased, therebyfurther decreasing leakage current and detector capacitance.

Signal Processing

The simplest noise approximation for the microluminometer assumes thedetection of a dc signal in wide band white noise. The input signal,x(t), may be approximated as a step function u(t), where the impulseresponse of the matched filter is:h _(opt)(t)=ku(t ₀ −t),  (38)and where k is a constant and to is the time of the measurement [28].The optimal impulse response has an output at negative infinity for animpulse input at t=0, and is therefore non-causal and non-realizable.However, the causal portion of the filter can be realized as a gatedintegrator with the gate open for 0<t<t₀ (FIG. 43).

The noise at the output a gated integrator due to white detector currentnoise at the input is: $\begin{matrix}{{\overset{\_}{v_{no}^{2}} = \frac{\overset{\_}{i_{n}^{2}}t_{0}}{2C_{f}^{2}}},} & (39)\end{matrix}$where

-   -   {overscore (ν² _(no))}=mean square output voltage noise    -   {overscore (i_(n) ²)}=mean square photodiode current noise        while C_(f)=integrator feedback capacitor, $\begin{matrix}        {{{v_{0}^{2}\left( t_{0} \right)} = \frac{i_{p}^{2}t_{0}^{2}}{C_{f}^{2}}},} & (40)        \end{matrix}$        where    -   ν_(o) ²(t_(o))=output signal power at t₀    -   i_(p)=photocurrent.

From equations (6) and (7) the signal-to-noise ratio (SNR) is$\begin{matrix}{{{SNR} = \frac{2i_{p}^{2}t_{0}}{\overset{\_}{i_{n}^{2}}}},} & (41)\end{matrix}$and continues to improve as t₀ increases.

Practical concerns will limit t₀ to several minutes. A remaining problemis capacitor values that are too large for on-chip implementation. Thiswas solved by using the hybrid analog/digital integration scheme asshown in FIG. 44. In this circuit, an analog integrator and adiscriminator convert the photodiode current into a train of digitalpulses (current-to-frequency converter (CFC)). These pulses are countedfor a fixed time (t₀), and the result is a digital word that isproportional to the photocurrent. This scheme has several advantagescompared to other processing options, including fast recovery fromoverload and ease of analog-to-digital conversion. It has been reportedas useful in optical detection systems (deGraff and Wolffenbuttel,1997).

Microluminometer Chip

FIG. 45 shows a photograph of a complete microluminometer chip. In thisexample, the chip measures 2.2 mm×2.2 mm with the photodetectoroccupying ˜25% (1.2 mm²) of the total chip area. For testing purposesthe chip was mounted in a 40-pin ceramic dual inline package.

All of the apparatus, compositions and methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the apparatus, compositions and methodsof this invention have been described in terms of preferred embodiments,it will be apparent to those of skill in the art that variations may beapplied to the apparatus, devices, methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents which are functionally related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. Accordingly, the exclusive rights sought to be patentedare as described in the claims below.

1. An integrated microluminometer, comprising: an integrated circuitchip that includes at least one n-well/p-substrate junctionphotodetector for converting light received into a photocurrent, and adetector on said chip for processing said photocurrent.
 2. Theintegrated microluminometer of claim 1, wherein said photodetectorincludes a distributed electrode configuration comprising a plurality ofspaced apart electrodes disposed on an active region of saidphotodetector.
 3. The integrated microluminometer of claim 1, whereinsaid detector comprises a current-to-frequency converter.
 4. Theintegrated microluminometer of claim 1, wherein said detector comprisesan analog gated integrator.
 5. The integrated microluminometer of claim4, further comprising a discriminator connected to an output of saidgated integrator, wherein said discriminator converts said photocurrentinto a train of digital pulses, said pulses being are counted for afixed time, wherein a digital word is generated which is proportional tosaid photocurrent.
 6. The integrated microluminometer of claim 5,wherein said discriminator comprises a comparator coupled to a one-shot,said one-shot coupled to a gated counter, wherein an output of saidgated counter is said digital word.
 7. An integrated microluminometer,comprising: a light detection system including at least one p-n junctionbased photodetector for converting light into electrical current andcircuitry for reducing or canceling recombination and generation currentproduced by said photodetector.
 8. The integrated microluminometer ofclaim 7, wherein said circuitry for reducing or canceling recombinationand generation current comprises at least one diode in a feedback loophaving an area smaller than an area of said photodetector, said diodesupplying forward bias current that cancels out photocurrent associatedwith said recombination and said generation current.
 9. The integratedmicroluminometer of claim 7, wherein said circuitry for reducing orcanceling recombination and generation current maintains said photodiodeat essentially zero bias.
 10. A method of measuring bioluminescence,comprising the steps of: providing the microluminometer of claim 3having a modified bioluminescent microorganism that emits light in thepresence of a selected analyte held on a surface of said chip;contacting said modified bioluminescent microorganism with a samplesuspected of containing said analyte, and operating saidmicroluminometer of claim 3 and counting light pulses produced for afixed time to determine said photocurrent, wherein said photocurrent isproportional to number of pulses that measure bioluminescence when saidanalyte causes said bioluminescent microorganism to emit said light. 11.The method of claim 10, wherein said modified bioluminescentmicroorganism is encapsulated in a sol-gel matrix held on said surface.